Treatment of kidney injury

ABSTRACT

Methods of treating and preventing kidney injury through inhibiting interleukin 11 (IL-11)-mediated signalling are disclosed, as well as agents for use in such methods.

This application claims priority from GB 1902419.9 filed 22 Feb. 2019,the contents and elements of which are herein incorporated by referencefor all purposes.

FIELD OF THE INVENTION

The present invention relates to the diagnosis, treatment andprophylaxis of diseases and conditions associated with kidney injury,particularly although not exclusively acute kidney injury.

BACKGROUND TO THE INVENTION

Acute kidney injury (AKI) refers to rapid onset damage to the kidney,notably the tubular epithelial cells. It is often chemically-driven, forexample renal damage or injury caused by a medicine, chemical, contrastdye or herbal or dietary supplements. It may also be caused byischaemia, mechanical or immune factors.

AKI is a common condition and affects up to 10% of patients in hospital(Silver, S. A. & Chertow, G. M. The Economic Consequences of AcuteKidney Injury. Nephron 137, 297-301 (2017)). There is major mortalityassociated with AKI and mortality in patients requiring intensive carepost AKI can be up to 50%. For those surviving AKI there are long-termrisks of progressing to chronic kidney disease (CKD), end stage renalfailure, renal replacement therapy or transplantation (Silver, S. A. &Chertow, G. M. The Economic Consequences of Acute Kidney Injury. Nephron137; Mehta, R. L et al. Acute Kidney Injury Network: report of aninitiative to improve outcomes in acute kidney injury. Crit. Care 11;Zuk, A. et al. Overcoming Translational Barriers in Acute Kidney Injury:A Report from an NIDDK Workshop. Clin. J. Am. Soc. Nephrol. 13,1113-1123 (2018)).

The chemotherapeutic agent cisplatin (dichlorodiamino platinum;SP-4-2)-diamminedichloroplatinum(II)) is widely used to treat a range ofcancers including head and neck, breast, lung, testis, ovarian, brain,and bladder cancers. After a single dose of cisplatin (50-100 mg/m²)most patients develop a degree of AKI and up to 30% of patients willdevelop nephrotoxicity (Ozkok, A. & Edelstein, C. L. Pathophysiology ofcisplatin-induced acute kidney injury. Biomed Res. Int. 2014, 967826(2014)), as defined by an abrupt reduction in kidney function signifiedby an increase in serum creatinine (Mehta et al supra). In elderlypatients with head and neck cancer up to 20% will progress from AKI tosevere kidney dysfunction (Yao, X., Panichpisal, K., Kurtzman, N. &Nugent, K. Cisplatin nephrotoxicity: a review. Am. J. Med. Sci. 334,115-124 (2007)). Cisplatin-induced AKI is dose limiting and therapy isoften split into multiple doses over several weeks but this is stillassociated with nephrotoxicity. The severity of cisplatin-induced AKI ismore severe in the presence of pre-existing conditions that includediabetes, hypertension, nephrotoxic drugs and old age.

The pathophysiology of AKI is largely defined by damage of the renaltubular epithelial cells (TECs) by toxins, chemicals, drugs, immune andmechanical factors or ischaemia. There are also vascular and immunecomponents to AKI. In patients treated with cisplatin, the drug ispresent in the kidney at levels up to five times those in the plasma. Inthe kidney, cisplatin accumulates in TECs where it induces reactiveoxygen species, depletes glutathione, causes mitochondrial dysfunctionand causes cell death through apoptosis or necrosis (Ozkok et al, supra;Wang, S., Wei, Q., Dong, G. & Dong, Z. ERK-mediated suppression of ciliain cisplatin-induced tubular cell apoptosis and acute kidney injury.Biochim. Biophys. Acta 1832, 1582-1590 (2013); Jo, S.-K., Cho, W. Y.,Sung, S. A., Kim, H. K. & Won, N. H. MEK inhibitor, U0126, attenuatescisplatin-induced renal injury by decreasing inflammation and apoptosis.Kidney Int. 67; Nowak, G. Protein Kinase C-α and ERK1/2 MediateMitochondrial Dysfunction, Decreases in Active Na+Transport, andCisplatin-induced Apoptosis in Renal Cells. J. Biol. Chem. 277,43377-43388 (2002)). During ischemia, TECs that have very high oxygendemands become oxygen deprived and undergo cell death due to apoptosisor necrosis.

Following AKI, kidney function can recover. This is widely recognised asdriven by proliferation of remaining TECs, which have very largeregenerative capacity (Yang. H.-C., Liu, S.-J. & Fogo, A. B. Kidneyregeneration in mammals. Nephron Exp. Nephrol. 126, 50 (2014); Coelho,S., Cabral, G., Lopes, J. A. & Jacinto, A. Renal regeneration afteracute kidney injury. Nephrology 23, 805-814 (2018); Chang-Panesso, M. &Humphreys, B. D. Cellular plasticity in kidney injury and repair. Nat.Rev. Nephrol. 13, 39-46 (2017)). When TEC proliferation is inadequatenephrotoxicity develops and over time chronic kidney disease can ensueand fibrosis may occur as a secondary phenomenon. In recent studies ithas been shown that a critical determinant of TEC dysfunction followingAKI is re-expression of the SNAIL gene (also known as SNA; SNAH; SNAIL;SLUGH2: SNAIL1) (Grande, M. T. et al. Snail1-induced partialepithelial-to-mesenchymal transition drives renal fibrosis in mice andcan be targeted to reverse established disease. Nat. Med. 21, 989-997(2015); Simon-Tillaux, N. & Hertig, A. Snail and kidney fibrosis.Nephrol. Dial. Transplant 32, 224-233 (2017); Lovisa, S. et al.Epithelial-to-mesenchymal transition induces cell cycle arrest andparenchymal damage in renal librosis. Nat. Med. 21, 998-1009 (2015)).SNAIL is important for embryogenesis but is rarely expressed in adultsother than in cancers where it causes epithelial to mesenchymaltransition (EMT). In the kidney, the TGFβ1 gene can induce SNAIL in TECsand this is associated with impaired TEC function and proliferation(Lovisa, S. et al. Epithelial-to-mesenchymal transition induces cellcycle arrest and parenchymal damage in renal fibrosis. Nat. Med. 21,998-1009 (2015)).

The cytokine interleukin 11 (IL-11) reportedly has a powerful protectiveeffect against AKI following ischemia-reperfusion injury and inhibitsapoptosis, necrosis and inflammation in AKI (Lee, H. T. et al.Interleukin-11 protects against renal ischemia and reperfusion injury.Am. J. Physiol. Renal Physiol. 303, F1216-24 (2012)). IL-11 has alsobeen shown to be upregulated in human TECs and mouse kidney byisoflurane and this is associated with a critical protective role forIL-11 against acute ischemic kidney injury (Ham, A. et al. Critical roleof interleukin-11 in isoflurane-mediated protection against ischemicacute kidney injury in mice. Anesthesiology 119, 1389-1401 (2013)).

IL-11 treatment has also been reported to protect against acutenephrotoxic nephritis in rats and mice by reducing kidney inflammationand preventing kidney damage (Stangou, M. et al. Effect of IL-11 onglomerular expression of TGF-beta and extracellular matrix innephrotoxic nephritis in Wistar Kyoto rats. J. Nephrol. 24, 106-111(2011): Lai, P. C. et al. Interleukin-11 attenuates nephrotoxicnephritis in Wistar Kyoto rats. J. Am. Soc. Nephrol. 12, 2310-2320(2001); Lai, P. C. et al. Interleukin-11 reduces renal injury andglomerular NF-kappa B activity in murine experimentalglomerulonephritis. Nephron Exp. Nephrol. 101, e146-54 (2005)).

SUMMARY OF THE INVENTION

In contrast to the reported protective role of IL-11 in kidney injuryand damage, the present invention relates to the treatment and/orprevention of kidney injury and disorders, diseases or conditionsassociated with kidney injury through the inhibition of IL-11signalling.

In one aspect of the present invention there is provided an agentcapable of inhibiting interleukin 11 (IL-11)-mediated signalling for usein a method of treating or preventing kidney injury and/or a disorder,disease or condition associated with kidney injury.

In another aspect of the present invention, there is provided the use ofan agent capable of inhibiting interleukin 11 (IL-11)-mediatedsignalling for use in the manufacture of a medicament for use in amethod of treating or preventing kidney injury and/or a disorder,disease or condition associated with kidney injury.

In another aspect of the present invention, there is provided a methodof treating or preventing kidney injury and/or a disorder, disease orcondition associated with kidney injury, the method comprisingadministering to a subject in need of treatment a therapeuticallyeffective amount of an agent capable of inhibiting interleukin 11(IL-11)-mediated signalling.

The present invention also provides an agent capable of inhibitinginterleukin 11 (IL-11)-mediated signalling for use in a method ofreversing kidney injury.

Also provided is the use of an agent capable of inhibiting interleukin11 (IL-11)-mediated signalling for use in the manufacture of amedicament for use in a method of reversing kidney injury.

Also provided is a method of reversing kidney injury, the methodcomprising administering to a subject in need of treatment atherapeutically effective amount of an agent capable of inhibitinginterleukin 11 (IL-11)-mediated signalling.

In some embodiments, the kidney injury is acute kidney injury. In someembodiments, the kidney injury is nephrotoxicity. In some embodiments,the kidney injury is drug-induced kidney injury or ischemia-inducedkidney injury. In some embodiments, the kidney injury iscisplatin-induced kidney injury or cisplatin-induced nephrotoxicity. Insome embodiments, the kidney injury is characterised by damage totubular epithelial cells (TECs).

The present invention also provides an agent capable of inhibitinginterleukin 11 (IL-11)-mediated signalling for use in a method ofimproving renal function in a subject suffering from an impairment torenal function.

Also provided is the use of an agent capable of inhibiting interleukin11 (IL-11)-mediated signalling for use in the manufacture of amedicament for use in a method of improving renal function in a subjectsuffering from an impairment to renal function.

Also provided is a method of improving renal function in a subjectsuffering from an impairment to renal function, the method comprisingadministering to a subject in need of treatment a therapeuticallyeffective amount of an agent capable of inhibiting interleukin 11(IL-11)-mediated signalling.

The present invention also provides an agent capable of inhibitinginterleukin 11 (IL-11)-mediated signalling for use in a method ofpromoting the proliferation, survival and/or function of tubularepithelial cells (TECs), and/or the growth, maintenance and/or functionof renal tissue (e.g. following kidney injury).

Also provided is the use of an agent capable of inhibiting interleukin11 (IL-11)-mediated signalling for use in the manufacture of amedicament for use in a method of promoting the proliferation, survivaland/or function of tubular epithelial cells (TECs), and/or the growth,maintenance and/or function of renal tissue (e.g. following kidneyinjury).

Also provided is a method of promoting the proliferation, survivaland/or function of tubular epithelial cells (TECs), and/or the growth,maintenance and/or function of renal tissue (e.g. following kidneyinjury), the method comprising administering to a subject in need oftreatment a therapeutically effective amount of an agent capable ofinhibiting interleukin 11 (IL-11)-mediated signalling.

The present invention also provides an agent capable of inhibitinginterleukin 11 (IL-11)-mediated signalling for use in a method ofinhibiting SNAIL expression (e.g. following kidney injury).

Also provided is the use of an agent capable of inhibiting interleukin11 (IL-11)-mediated signalling for use in the manufacture of amedicament for use in a method of inhibiting SNAIL expression (e.g.following kidney injury).

Also provided is a method of inhibiting SNAIL expression (e.g. followingkidney injury), the method comprising administering to a subject in needof treatment a therapeutically effective amount of an agent capable ofinhibiting interleukin 11 (IL-11)-mediated signalling.

The present invention also provides an agent capable of inhibitinginterleukin 11 (IL-11)-mediated signalling for use in a method ofinhibiting or reversing the transition of tubular epithelial cells(TECs) to a mesenchymal cell-like phenotype (e.g. following kidneyinjury).

Also provided is the use of an agent capable of inhibiting interleukin11 (IL-11)-mediated signalling for use in the manufacture of amedicament for use in a method of inhibiting or reversing the transitionof tubular epithelial cells (TECs) to a mesenchymal cell-like phenotype(e.g. following kidney injury).

Also provided is a method of inhibiting or reversing the transition oftubular epithelial cells (TECs) to a mesenchymal cell-like phenotype(e.g. following kidney injury), the method comprising administering to asubject in need of treatment a therapeutically effective amount of anagent capable of inhibiting interleukin 11 (IL-11)-mediated signalling.

In some embodiments in accordance with various aspects of the presentinvention, the agent is an agent capable of preventing or reducing thebinding of interleukin 11 (IL-11) to a receptor for interleukin 11(IL-11R).

In some embodiments, the agent is capable of binding to interleukin 11(IL-11) or a receptor for interleukin 11 (IL-11R). In some embodiments,the agent is selected from the group consisting of: an antibody or anantigen-binding fragment thereof, a polypeptide, a peptide, a nucleicacid, an oligonucleotide, an aptamer or a small molecule. The agent maybe an antibody or an antigen-binding fragment thereof. The agent may bea decoy receptor.

In some embodiments, the agent is an anti-IL-11 antibody antagonist ofIL-11-mediated signalling, or an antigen-binding fragment thereof.

In some embodiments, the antibody or antigen-binding fragment comprises:

(i) a heavy chain variable (VH) region incorporating the following CDRs:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:34    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:35    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:36; and

(ii) a light chain variable (VL) region incorporating the followingCDRs:

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:37    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:38    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:39.

In some embodiments, the antibody or antigen-binding fragment comprises:

(i) a heavy chain variable (VH) region incorporating the following CDRs:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:40    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:41    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:42; and

(ii) a light chain variable (VL) region incorporating the followingCDRs:

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:43    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:44    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:45.

In some embodiments, the agent is an anti-IL-11Rα antibody antagonist ofIL-11-mediated signalling, or an antigen-binding fragment thereof.

In some embodiments, the antibody or antigen-binding fragment comprises:

(i) a heavy chain variable (VH) region incorporating the following CDRs:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:46    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:47    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48; and

(ii) a light chain variable (VL) region incorporating the followingCDRs:

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:49    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:50    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:51.

In some embodiments, the agent is a decoy receptor for IL-11. In someembodiments the decoy receptor for IL-11 comprises: (i) an amino acidsequence corresponding to the cytokine binding module of gp130 and (ii)an amino acid sequence corresponding to the cytokine binding module ofIL-11Rα.

In some embodiments the agent is an IL-11 mutein. In some embodimentsthe IL-11 mutein is W147A.

In some embodiments, the agent is capable of preventing or reducing theexpression of interleukin 11 (IL-11) or a receptor for interleukin 11(IL-11R). The agent may be an oligonucleotide or a small molecule.

In some embodiments the agent is an antisense oligonucleotide capable ofpreventing or reducing the expression of IL-11. In some embodiments theantisense oligonucleotide capable of preventing or reducing theexpression of IL-11 is siRNA targeted to IL11 comprising the sequence ofSEQ ID NO:12, 13, 14 or 15.

In some embodiments the agent is an antisense oligonucleotide capable ofpreventing or reducing the expression of IL-11Rα. In some embodimentsthe antisense oligonucleotide capable of preventing or reducing theexpression of IL-11Rα is siRNA targeted to IL11RA comprising thesequence of SEQ ID NO:16, 17, 18 or 19.

In any embodiments provided herein, the interleukin 11 receptor may beor comprise IL-11Rα.

In any embodiments provided herein, the kidney injury may be one ofacute kidney injury (AKI), nephrotoxicity, drug-induced kidney injury(DIKI), acute kidney failure, acute kidney disease, chronic kidneydisease, kidney damage, tubular necrosis, acute tubular necrosis, andautoimmune kidney injury.

In any embodiments provided herein, the agent may be administeredbefore, in conjunction with, or after the cause of the kidney injury,e.g. administration or consumption of a nephrotoxic medicine or exposureto a physical, mechanical, chemical or environmental source of kidneyinjury.

In some embodiments, the agents, uses and methods herein are providedfor treating and/or preventing drug-induced kidney injury (DIKI). TheDIKI may be intrinsic and/or idiosyncratic kidney injury. In anyembodiments, the agents, uses and methods herein may be provided fortreating and/or preventing cisplatin-induced kidney injury and/orcisplatin-induced nephrotoxicity.

In some embodiments, the agents, uses and methods herein may be providedfor treating and/or preventing ischemia-induced kidney injury (IIKI) orischemia-induced acute kidney injury.

In any embodiments provided herein, the disorder, disease or conditionassociated with kidney injury may be a disease, disorder or condition inwhich kidney injury is pathologically implicated. The pathology mayinclude damage to tubular epithelial cells and/or the transition of TECsto a mesenchymal cell-Ike phenotype, which may be proximal and/ordistal.

In any embodiments, the method comprises administering the agent to asubject in which expression of interleukin 11 (IL-11) or a receptor forIL-11 (IL-11R) may be upregulated.

In any embodiments, the method may comprise administering the agent to asubject in which expression of interleukin 11 (IL-11) or a receptor forinterleukin 11 (IL-11R) has been determined to be upregulated.

In some embodiments the method comprises determining whether expressionof interleukin 11 (IL-11) or a receptor for IL-11 (IL-11R) isupregulated in the subject and administering the agent to a subject inwhich expression of interleukin 11 (IL-11) or a receptor for IL-11(IL-11R) is upregulated.

Also provided is a method of determining the suitability of a subjectfor the treatment or prevention of kidney injury and/or a disorder,disease or condition associated with kidney injury with an agent capableof inhibiting interleukin 11 (IL-11)-mediated signalling, the methodcomprising determining, optionally in vitro, whether interleukin 11(IL-11) or a receptor for IL-11 (IL-11R) expression is upregulated inthe subject.

Also provided is a method of selecting a subject for the treatment orprevention of kidney injury and/or a disorder, disease or conditionassociated with kidney injury with an agent capable of inhibitinginterleukin 11 (IL-11)-mediated signalling, the method comprisingdetermining, optionally in vitro, whether interleukin 11 (IL-11) or areceptor for IL-11 (IL-11R) expression is upregulated in the subject.

In one aspect there is provided a method of diagnosing kidney injuryand/or a disorder, disease or condition associated with kidney injury,or a risk of developing kidney injury and/or a disorder, disease orcondition associated with kidney injury in a subject, the methodcomprising determining, optionally in vitro, the upregulation ofinterleukin 11 (IL-11) or an receptor for IL-11 (IL-11R) in a sampleobtained from the subject. In some embodiments, the method of diagnosingis a method of confirming a diagnosis of kidney injury and/or adisorder, disease or condition associated with kidney injury in asubject suspected of having kidney injury and/or a disorder, disease orcondition associated with kidney injury. In some embodiments a method ofdiagnosing and/or a method of confirming a diagnosis comprises selectingthe subject for treatment with an agent capable of inhibitinginterleukin 11 (IL-11)-mediated signalling.

Also provided is a method of providing a prognosis for a subject having,or suspected of having, kidney injury and/or a disorder, disease orcondition associated with kidney injury, the method comprisingdetermining, optionally in vitro, whether expression of interleukin 11(IL-11) or a receptor for IL-11 (IL-11R) is upregulated in a sampleobtained from the subject and, based on the determination, providing aprognosis for treatment of the subject with an agent capable ofinhibiting interleukin 11 (IL-11)-mediated signalling. In someembodiments, a method of providing a prognosis comprises selecting asubject determined to have upregulated expression of interleukin 11(IL-11) or a receptor for IL-11 (IL-11R) for treatment with an agentcapable of inhibiting interleukin 11 (IL-11)-mediated signalling.

In another aspect there is provided a method of diagnosing kidney injuryand/or a disorder, disease or condition associated with kidney injury ora risk of developing kidney injury and/or a disorder, disease orcondition associated with kidney injury, the method comprisingdetermining, optionally in vitro, one or more genetic factors in thesubject that are predictive of upregulation of expression of IL-11 or areceptor for IL-11, or of upregulation of IL-11 mediated signalling. Insome embodiments the method comprises selecting the subject fortreatment with an agent capable of inhibiting interleukin 11(IL-11)-mediated signalling.

Also provided is a method of providing a prognosis for a subject having,or suspected of having, kidney injury and/or a disorder, disease orcondition associated with kidney injury, the method comprisingdetermining, optionally in vitro, one or more genetic factors in thesubject that are predictive of upregulation of expression of IL-11 or areceptor for IL-11, or of upregulation of IL-11 mediated signalling.

DESCRIPTION

There is ongoing demand for effective prevention and treatment of kidneyinjury, particularly acute kidney injury.

The cytokine IL-11 has been reported to have a protective effect againstacute kidney injury following ischemia-reperfusion injury (Lee et alsupra) and IL-11 has been reported to be upregulated and protective inhuman tubular epithelial cells and mouse kidney upon chemical damage byisoflurane (Ham et al supra).

In contrast, however, the present inventors have found that inhibitionof IL-11 mediated signalling is effective to protect tubular epithelialcells, allowing them to proliferate and recover from damage that is acommon causative factor of acute kidney injury. Whilst not wishing to bebound by theory, the inventors believe that inhibition of IL-11 enablestubular epithelial cells to proliferate leading to renal tissueregeneration and recovery by preventing or reducing the expression ofSNAIL.

Interleukin 11 and Receptors for IL-11

Interleukin 11 (IL-11), also known as adipogenesis inhibitory factor, isa pleiotropic cytokine and a member of the IL-6 family of cytokines thatincludes IL-6, IL-11, IL-27, IL-31, oncostatin, leukemia inhibitoryfactor (LIF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC),ciliary neurotrophic factor (CNTF) and neuropoetin (NP-1).

Interleukin 11 (IL-11) is expressed in a variety of mesenchymal celltypes. IL-11 genomic sequences have been mapped onto chromosome 19 andthe centromeric region of chromosome 7, and is transcribed with acanonical signal peptide that ensures efficient secretion from cells.The activator protein complex of IL-11, cJun/AP-1, located within itspromoter sequence is critical for basal transcriptional regulation ofIL-11 (Du and Williams., Blood 1997, Vol 89: 3897-3908). The immatureform of human IL-11 is a 199 amino acid polypeptide whereas the matureform of IL-11 encodes a protein of 178 amino acid residues (Garbers andScheller., Biol. Chem. 2013; 394(9):1145-1161). The human IL-11 aminoacid sequence is available under UniProt accession no. P20809 (P20809.1GI:124294; SEQ ID NO:1). Recombinant human IL-11 (oprelvekin) is alsocommercially available. IL-11 from other species, including mouse, rat,pig, cow, several species of bony fish and primates, have also beencloned and sequenced.

In this specification “IL-11” refers to an IL-11 from any species andincludes isoforms, fragments, variants or homologues of an IL-11 fromany species. In preferred embodiments the species is human (Homosapiens). Isoforms, fragments, variants or homologues of an IL-11 mayoptionally be characterised as having at least 70%, preferably one of80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% aminoacid sequence identity to the amino acid sequence of immature or matureIL-11 from a given species, e.g. human. Isoforms, fragments, variants orhomologues of an IL-11 may optionally be characterised by ability tobind IL-11Rα (preferably from the same species) and stimulate signaltransduction in cells expressing IL-11Rα and gp130 (e.g. as described inCurtis et al. Blood, 1997, 90(11); or Karpovich et al. Mol. Hum. Reprod.2003 9(2): 75-80). A fragment of IL-11 may be of any length (by numberof amino acids), although may optionally be at least 25% of the lengthof mature IL-11 and may have a maximum length of one of 50%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the lengthof mature IL-11. A fragment of IL-11 may have a minimum length of 10amino acids, and a maximum length of one of 15, 20, 25, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 195amino acids.

IL-11 signals through a homodimer of the ubiquitously expressedglycoprotein 130 (gp130; also known as glycoprotein 130, IL-6ST,IL-6-beta or CD130). Gp130 is a transmembrane protein that forms onesubunit of the type I cytokine receptor with the IL-6 receptor family.Specificity is gained through an individual interleukin 11 receptorsubunit alpha (IL-11Rα), which does not directly participate in signaltransduction, although the initial cytokine binding event to theα-receptor leads to the final complex formation with gp130.

Human gp130 (including the 22 amino acid signal peptide) is a 918 aminoacid protein, and the mature form is 866 amino acids, comprising a 597amino acid extracellular domain, a 22 amino acid transmembrane domain,and a 277 amino acid intracellular domain. The extracellular domain ofthe protein comprises the cytokine-binding module (CBM) of gp130. TheCBM of gp130 comprises the Ig-like domain D1, and the fibronectin-typeIII domains D2 and D3 of gp130. The amino acid sequence of human gp130is available under UniProt accession no. P40189-1 (SEQ ID NO:2).

Human IL-11Rα is a 422 amino acid polypeptide (UniProt 014626; SEQ IDNO:3) and shares ˜85% nucleotide and amino acid sequence identity withthe murine IL-11Rα. Two isoforms of IL-11Rα have been reported, whichdiffer in the cytoplasmic domain (Du and Williams, supra). The IL-11receptor α-chain (IL-11Rα) shares many structural and functionalsimilarities with the IL-6 receptor α-chain (IL-6Rα). The extracellulardomain shows 24% amino acid identity including the characteristicconserved Trp-Ser-X-Trp-Ser (WSXWS) motif. The short cytoplasmic domain(34 amino acids) lacks the Box 1 and 2 regions that are required foractivation of the JAK/STAT signalling pathway.

The receptor binding sites on murine IL-11 have been mapped and threesites—sites I, II and III—identified. Binding to gp130 is reduced bysubstitutions in the site II region and by substitutions in the site IIIregion. Site III mutants show no detectable agonist activity and haveIL-11Rα antagonist activity (Cytokine Inhibitors Chapter 8; edited byGennaro Ciliberto and Rocco Savino, Marcel Dekker, Inc. 2001).

In this specification a receptor for IL-11 (IL-11R) refers to apolypeptide or polypeptide complex capable of binding IL-11. In someembodiments an IL-11 receptor is capable of binding IL-11 and inducingsignal transduction in cells expressing the receptor.

An IL-11 receptor may be from any species and includes isoforms,fragments, variants or homologues of an IL-11 receptor from any species.In preferred embodiments the species is human (Homo sapiens).

In some embodiments the IL-11 receptor may be IL-11Rα. In someembodiments a receptor for IL-11 may be a polypeptide complex comprisingIL-11Rα. In some embodiments the IL-11 receptor may be a polypeptidecomplex comprising IL-11Rα and gp130. In some embodiments the IL-11receptor may be gp130 or a complex comprising gp130 to which IL-11binds.

Isoforms, fragments, variants or homologues of an IL-11Rα may optionallybe characterised as having at least 70%, preferably one of 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity to the amino acid sequence of IL-11Rα from a givenspecies, e.g. human. Isoforms, fragments, variants or homologues of anIL-11Rα may optionally be characterised by ability to bind IL-11(preferably from the same species) and stimulate signal transduction incells expressing the IL-11Rα and gp130 (e.g. as described in Curtis etal. Blood, 1997, 90(11) or Karpovich et al. Mol. Hum. Reprod. 2003 9(2):75-80). A fragment of an IL-11 receptor may be of any length (by numberof amino acids), although may optionally be at least 25% of the lengthof the mature IL-11Rα and have a maximum length of one of 50%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of the lengthof the mature IL-11Rα. A fragment of an IL-11 receptor fragment may havea minimum length of 10 amino acids, and a maximum length of one of 15,20, 25, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 250, 300, 400, or 415 amino acids.

IL-11 Signalling

IL-11 binds to IL-11Rα with low affinity (Kd ˜10 nmol/L), andinteraction between these binding partners alone is insufficient totransduce a biological signal. The generation of a high affinityreceptor (Kd ˜400 to 800 pmol/L) capable of signal transduction requiresco-expression of the IL-11Rα and gp130 (Curtis et al Blood 1997; 90(11):4403-12; Hilton et al., EMBO J 13:4765, 1994; Nandurkar et al.,Oncogene 12:585, 1996). Binding of IL-11 to cell-surface IL-11Rα inducesheterodimerization, tyrosine phosphorylation, activation of gp130 anddownstream signalling, predominantly through the mitogen-activatedprotein kinase (MAPK)-cascade and the Janus kinase/signal transducer andactivator of transcription (Jak/STAT) pathway (Garbers and Scheller,supra).

In principle, a soluble IL-11Rα can also form biologically activesoluble complexes with IL-11 (Pflanz et al., 1999 FEBS Lett, 450,117-122) raising the possibility that, similar to IL-6, IL-11 may insome instances bind soluble IL-11Rα prior to binding cell-surface gp130(Garbers and Scheller, supra). Curtis et al (Blood 1997 Dec. 1; 90(11):4403-12) describe expression of a soluble murine IL-11 receptoralpha chain (sIL-11R) and examined signalling in cells expressing gp130.In the presence of gp130 but not transmembrane IL-11R the sIL-11Rmediated IL-11 dependent differentiation of M1 leukemic cells andproliferation in Ba/F3 cells and early intracellular events includingphosphorylation of gp130, STAT3 and SHP2 similar to signalling throughtransmembrane IL-11R. Activation of signalling through cell-membranebound gp130 by IL-11 bound to soluble IL-11Rα has recently beendemonstrated (Lokau et al., 2016 Cell Reports 14, 1761-1773). Thisso-called IL-11 trans signalling may be important for diseasepathogenesis, yet its role in human disease has not yet been studied. Inpreferred embodiments, inhibition of IL-11-mediated signalling isachieved by disrupting IL-11-mediated cis signalling.

As used herein. ‘IL-11 trans signalling’ is used to refer to signallingwhich is triggered by binding of IL-11 bound to IL-11Rα, to gp130. TheIL-11 may be bound to IL-11Rα as a non-covalent complex. The gp130 ismembrane-bound and expressed by the cell in which signalling occursfollowing binding of the IL-11:IL-11Rα complex to gp130. In someembodiments the IL-11Rα may be a soluble IL-11Rα. In some embodiments,the soluble IL-11Rα is a soluble (secreted) isoform of IL-11Rα (e.g.lacking a transmembrane domain). In some embodiments, the solubleIL-11Rα is the liberated product of proteolytic cleavage of theextracellular domain of cell membrane bound IL-11Rα. In someembodiments, the IL-11Rα may be cell membrane-bound, and signallingthrough gp130 may be triggered by binding of IL-11 bound tocell-membrane-bound IL-11Rα, termed “IL-11 cis signalling”.

IL-11-mediated signalling has been shown to stimulate hematopoiesis andthrombopoiesis, stimulate osteoclast activity, stimulate neurogenesis,inhibit adipogenesis, reduce pro inflammatory cytokine expression,modulate extracellular matrix (ECM) metabolism, and mediate normalgrowth control of gastrointestinal epithelial cells (Du and Williams,supra).

The physiological role of Interleukin 11 (IL-11) remains unclear. IL-11has been most strongly linked with activation of haematopoietic cellsand with platelet production. IL-11 has also been shown to conferprotection against graft-vs-host-disease, inflammatory arthritis andinflammatory bowel disease, leading to IL-11 being considered ananti-inflammatory cytokine (Putoczki and Ernst, J Leukoc Biol 2010,88(6):1109-1117). However, it is suggested that IL-11 ispro-inflammatory as well as anti-inflammatory, pro-angiogenic andimportant for neoplasia. Recent studies have shown that IL-11 is readilydetectable during viral-induced inflammation in a mouse arthritis modeland in cancers, suggesting that the expression of IL-11 can be inducedby pathological stimuli. IL-11 is also linked to Stat3-dependentactivation of tumour-promoting target genes in neoplasticgastrointestinal epithelium (Putoczki and Ernst, supra).

As used herein, “IL-11 signalling” and “IL-11-mediated signalling”refers to signalling mediated by binding of IL-11, or a fragment thereofhaving the function of the mature IL-11 molecule, to a receptor forIL-11. It will be appreciated that “IL-11 signalling” and “IL-11mediated signalling” refer to signalling initiated by IL-11/functionalfragment thereof, e.g. through binding to a receptor for IL-11.“Signalling” in turn refers to signal transduction and other cellularprocesses governing cellular activity.

Kidney Injury

Aspects of the present invention relate to the diagnosis, treatment andprophylaxis of kidney injury, particularly acute kidney injury (AKI;also known as acute renal failure) and/or kidney injury, e.g. AKI.

Herein, ‘kidney injury’ refers to damage to the kidney, renal tissue,and/or one or more renal cells. Damage to a cell/tissue/organ may resultfrom insult to the cell/tissue/organ, e.g. chemical or physicaltreatment/experience. In some embodiments kidney injury may be aconsequence of chemical insult, e.g. in the case of drug-induced kidneyinjury, e.g. cisplatin-induced kidney injury. In some embodiments kidneyinjury may arise from physical insult, e.g. in the case of kidney injuryarising as a result of crush, or kidney injury as a result of surgicaldamage to renal tissue, which may occur e.g. during surgery to treat adisease and/or for kidney transplantation (e.g. the kidney injury mayhave iatrogenic causes). In some embodiments kidney injury may be aconsequence of hypoxia, e.g. as a consequence of ischaemia, or mayresult from reperfusion. In some embodiments kidney injury may arisefrom infection, immune response to infection, cancer and/orautoimmunity. Damage may be reversible or irreversible.

In some embodiments, kidney injury comprises damage to one or both ofthe kidneys. In some embodiments kidney injury comprises damage to oneor more of a renal capsule, renal cortex, renal medulla, renal papilla,renal pyramid, renal column, renal calyx, minor calyx, major calyx,hilum, renal pelvis, ureter, renal artery and a renal vein. In someembodiments kidney injury comprises damage to one or more of a renalepithelial cell, a tubule epithelial cell, a proximal tubule epithelialcell, a distal tubule epithelial cell, a renal parietal cell, apodocyte, a loop of Henle thin segment cell, a thick ascending limbcell, a collecting duct principal cell, a collecting duct intercalatedcell, and an interstitial kidney cell. In some embodiments, kidneyinjury comprises damage to a renal epithelial cell, e.g. a tubuleepithelial cell, e.g. proximal tubule epithelial cell.

Damage to a cell/tissue/organ may be characterised by a change to thestructure and/or function of the cell/tissue/organ. For example, damageto a cell/tissue/organ may be characterised by a reduction in the levelof a correlate of normal function of the cell/tissue/organ, and/or anincrease in a correlate of impaired function of the cell/tissue/organ.By way of illustration, damage to the kidney/renal tissue/renal cellsmay be characterised by a reduction in urine output by the subjectexperiencing kidney damage, and/or an increase in serum creatine levelsin the subject experiencing kidney damage. Damage to a cell/tissue/organmay be characterised by cell death, e.g. death of cells of the damagedorgan/tissue. The cell death may result from apoptosis (i.e. programmedcell death) or necrosis (premature cell death as a consequence ofdamage).

As used herein, ‘acute kidney injury’ generally refers to abruptdeterioration in kidney function. Acute kidney injury may becharacterised by a rapid increase serum creatinine and/or a rapidreduction in urine output.

Acute kidney injury may be defined and staged in accordance with theSummary of Recommendation Statements of the Kidney Disease ImprovingGlobal Outcomes (KDIGO) group, Kidney International Supplements (2012)2, 8-12 (hereby incorporated by reference in its entirety).

In some embodiments, acute kidney injury is defined as: (i) an increasein serum creatine by ≥0.3 mg/dl (≥26.5 μmol/l) within a 48 hour period;(ii) an increase in serum creatine to ≥1.5 times baseline which is knownor presumed to have occurred within the previous 7 days: or (iii) urinevolume of <0.5 ml/kg/h for 6 hours.

In some embodiments, the acute kidney injury may be stage 1, 2 or 3acute kidney injury. Stage 1 acute kidney injury may be defined as: (i)an increase in serum creatine to 1.5-1.9 times baseline, (ii) anincrease in serum creatine to ≥0.3 mg/dl (≥26.5 μmol/l), or (iii) urinevolume of <0.5 ml/kg/h for 6-12 hours. Stage 2 acute kidney injury maybe defined as: (i) an increase in serum creatine to 2.0-2.9 timesbaseline, or (ii) urine volume of <0.5 ml/kg/h for 212 hours. Stage 3acute kidney injury may be defined as: (i) an increase in serum creatineto ≥3.0 times baseline, (ii) an increase in serum creatine to ≥4.0 mg/dl(≥353.6 μmol/l), (iii) initiation of renal replacement therapy, (iv) inpateints <18 years, decrease in eGFR to <35 ml/min per 1.73 m²; (v)urine volume of <0.3 mV/kg/h for 224 hours, or (vi) anuria for 212hours.

Kidney injury may be characterised by damage to tubular epithelial cells(TECs) and/or the transition of TECs to an epithelial-to-mesenchymalcell-like phenotype (i.e. EMT). Transition of TECs to a mesenchymalcell-like phenotype may be characterised e.g. by reduced expression ofE-cadherin, increased expression of SNAIL and/or increased expression ofACTA2.

The kidney injury may have any cause, examples include kidney injuryresulting from mechanical (i.e. physical) damage or injury, chemicaldamage or injury, ischemia or genetic predisposition. The cause ordamage will normally result in impaired kidney function, which may leadto kidney failure.

Mechanical damage or injury may include physical injury to the subject,to the kidney, to TECs or to podocytes. It may also include tubularobstruction/blockage, e.g. of the urinary tract.

Chemical damage or injury may be caused by a drug, medicine, toxin,herbal or dietary supplements or other chemical agent administered to,absorbed or ingested by the subject. In some embodiments the chemicaldamage is a side effect of the administration of such an agent to treata disease or condition not occurring in the kidney, or occurring both inthe kidney and in one or more other tissues. In some embodiments thechemical damage is a side effect of administration of a chemotherapeuticagent administered to the subject in order to prevent or treat cancer.In some embodiments the kidney injury is drug-induced kidney injury ordrug-induced acute kidney injury. In some embodiments, tubularobstruction/blockage, e.g. of the urinary tract, may arise as the resultof administration of certain chemical agents, e.g. sulphonamides,methotrexate, acyclovir, diethylene glycol, triamterene.

Ischemic damage may arise from a decrease in blood flow to the kidneywhich may be caused by a number of factors such as low blood pressuree.g. due to sepsis, blood loss or surgery, or the effect of a chemicalagent, e.g. a medicine or drug, administered to the subject to treatanother disease, disorder or condition. Kidney injury caused by ischemiamay be ischemia-induced kidney injury, or ischemia-induced acute kidneyinjury. Kidney injury caused by crush injury may be ischemia-inducedkidney injury with vasoconstriction or can be caused by tubular castmechanical factors or toxic effects of circulating factors e.g.myoglobin.

In some embodiments the kidney injury, which may be AKI, ischaracterised by damage to, which may in some cases include or lead todeath of, tubular epithelial cells (TECs) of the kidney, i.e. renaltubular epithelial cells. The TECs may be proximal or distal, both ofwhich may be damaged in AKI, as may also the podocytes in the kidneyglomerulus. Damage to TECs may also be any type of damage, injury orinsult, e.g. as described above this may be mechanical, chemical orischemic damage. Damage to TECs is a common causative factor of kidneyinjury, particularly AKI. Proliferation of TECs provides a mechanism forrecovery and restoration of kidney function, whereas failure of TECs toproliferate can lead to disease development and progression, e.g. tochronic kidney disease and renal failure. Proliferation of podocyteprecursors to restore glomerulus function may also occur, but is not aswell described as TEC proliferation.

In some embodiments the kidney injury is, or is characterised by,nephrotoxicity. As used herein, nephrotoxicity refers to toxicity to thekidneys. Toxicity in turn refers to damage, e.g. as described herein.Nephrotoxicity can arise as a result of toxic effects of certainsubstances on renal function, and may therefore be viewed as aconsequence of chemical damage or injury. As with chemical damage orinjury, nephrotoxicity may be a side effect of the administration of anagent to treat a disease or condition not occurring in the kidney, oroccurring both in the kidney and in one or more other tissues. In someembodiments nephrotoxicity may be a side effect of administration of achemotherapeutic agent administered to the subject in order to preventor treat cancer. As such, nephrotoxicity may be a form of drug-inducedkidney injury or drug-induced acute kidney injury.

As described above, drug-induced kidney injury, drug-induced acutekidney injury or drug-induced nephrotoxicity may arise as a side effectof the administration of a drug or medicine intended to treat a disease,disorder or condition occurring in tissues outside the kidney, withinthe kidney or both. A number of drugs are known to exhibit suchside-effects, which may depend on the condition being treated, thesubject being treated, the dosage amount, dosage regime or mode ofadministration, and the extent to which the subject exhibits partialkidney failure prior to treatment. For example, the following agentshave all been reported to have potential in inducing kidney injury:diuretics, n-blockers, vasodilators, ACE inhibitors, aminoglycosideantibiotics (e.g. gentamicin), amphotericin B, cisplatin, NSAIDs (e.g.aspirin, ibuprofen, diclofenac), ciclosporin, lithium salts,cyclophosphamide, sulphonamides, methotrexate, acyclovir, diethyleneglycol, triamterene, β-lactam antibiotics, vancomycin, rilampicin,ciprofloxacin, ranitidine, cimetidine, furosemide, thiazides, phenytoin.Optionally a drug-induced kidney injury is not a folate-induced kidneyinjury.

In some embodiments, a drug-induced kidney injury, drug-induced acutekidney injury or drug-induced nephrotoxicity may arise as a side effectof the administration of a chemotherapeutic agent intended to treat orprevent cancer in the subject. Examples of chemotherapeutic agentsinclude alkylating agents such as cisplatin, carboplatin,mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; purine orpyrimidine anti-metabolites such as azathiopurine or mercaptopurine;alkaloids and terpenoids, such as vinca alkaloids (e.g. vincristine,vinblastine, vinorelbine, vindesine), podophyllotoxin, etoposide,teniposide, taxanes such as paclitaxel (Taxol™), docetaxel;topoisomerase inhibitors such as the type I topoisomerase inhibitorscamptothecins irinotecan and topotecan, or the type II topoisomeraseinhibitors amsacrine, etoposide, etoposide phosphate, teniposide;antitumor antibiotics (e.g. anthracycline antibiotics) such asdactinomycin, doxorubicin (Adriamycin™), epirubicin, bleomycin,rapamycin; antibody based agents, such as anti-VEGF, anti-TNFα,anti-IL-2, antiGpIIb/IIIa, anti-CD-52, anti-CD20, anti-RSV,anti-HER2/neu(erbB2), anti-TNF receptor, anti-EGFR antibodies,monoclonal antibodies or antibody fragments, examples include:cetuximab, panitumumab, infliximab, basiliximab, bevacizumab (Avastin®),abciximab, daclizumab, gemtuzumab, alemtuzumab, rituximab (Mabthera®),palivizumab, trastuzumab, etanercept, adalimumab, nimotuzumab; and EGFRinhibitors such as erlotinib, cetuximab, gefitinib.

In some preferred embodiments, the present invention concerns thediagnosis, treatment and/or prophylaxis of cisplatin-induced kidneyinjury. This may include cisplatin-induced acute kidney injury orcisplatin-induced nephrotoxicity. Cisplatin (dichlorodiamino platinum;SP-4-2)-diamminedichloroplatinum(II)) is a chemotherapeutic agent thatis widely used to treat a range of cancers including head and neck,breast, lung, testis, ovarian, brain, and bladder cancers and is widelyacknowledged to lead to kidney injury and dysfunction involving tubulardamage and necrosis (e.g. Oh et al., Electrolyte Blood Press 2014December; 12(2): 55-65; P A Arunkumar et al., Asian Pac J Trop Biomed2012 Aug. 2(8): 640-644). Other platinum-based chemotherapeutics agentsalso cause kidney damage.

Whilst it is recognised that a subject having kidney injury may alsopresent with fibrosis of the kidney, either as a disease conditionhaving a separable etiology or as a secondary effect of the kidneyinjury, in some embodiments the kidney injury being diagnosed, treatedor prevented is not librosis of the kidney, e.g. renal fibrosis. In someembodiments the subject does not have fibrosis. In some embodiments TECdamage occurs in the absence of fibrosis. In some embodiments fibrosisarises separately (e.g. secondarily to) AKI, e.g. due to incompleteregeneration of TECs. In some embodiments, the damaged TECs in thesubject are not pro-fibrotic TECs. In some embodiments, fibrosis doesnot arise.

Agents Capable of Inhibiting the Action of IL-11

Aspects of the present invention involve inhibition of IL-11-mediatedsignalling.

Herein, ‘inhibition’ refers to a reduction, decrease or lesseningrelative to a control condition. For example, inhibition of the actionof IL-11 by an agent capable of inhibiting IL-11-mediated signallingrefers to a reduction, decrease or lessening of the extent/degree ofIL-11-mediated signalling in the absence of the agent, and/or in thepresence of an appropriate control agent.

Inhibition may herein also be referred to as neutralisation orantagonism. That is, an agent capable of inhibiting IL-11-mediatedsignalling (e.g. interaction, signalling or other activity mediated byIL-11 or an IL-11-containing complex) may be said to be a ‘neutralising’or ‘antagonist’ agent with respect to the relevant function or process.For example, an agent which is capable of inhibiting IL-11-mediatedsignalling may be referred to as an agent which is capable ofneutralising IL-11-mediated signalling, or may be referred to as anantagonist of IL-11-mediated signalling.

The IL-11 signalling pathway offers multiple routes for inhibition ofIL-11 signalling. An agent capable of inhibiting IL-11-mediatedsignalling may do so e.g. through inhibiting the action of one or morefactors involved in, or necessary for, signalling through a receptor forIL-11.

For example, inhibition of IL-11 signalling may be achieved bydisrupting interaction between IL-11 (or an IL-11 containing complex,e.g. a complex of IL-11 and IL-11Rα) and a receptor for IL-11 (e.g.IL-11Rα, a receptor complex comprising IL-11Rα, gp130 or a receptorcomplex comprising IL-11Rα and gp130). In some embodiments, inhibitionof IL-11-mediated signalling is achieved by inhibiting the gene orprotein expression of one or more of e.g. IL-11, IL-11Rα and gp130.

In embodiments, inhibition of IL-11-mediated signalling is achieved bydisrupting IL-11-mediated cis signalling but not disruptingIL-11-mediated trans signalling, e.g. inhibition of IL-11-mediatedsignalling is achieved by inhibiting gp130-mediated cis complexesinvolving membrane bound IL-11Rα. In embodiments, inhibition ofIL-11-mediated signalling is achieved by disrupting IL-11-mediated transsignalling but not disrupting IL-11-mediated cis signalling, i.e.inhibition of IL-11-mediated signalling is achieved by inhibitinggp130-mediated trans signalling complexes such as IL-11 bound to solubleIL-11Rα or IL-6 bound to soluble IL-6R. In embodiments, inhibition ofIL-11-mediated signalling is achieved by disrupting IL-11-mediated cissignalling and IL-11-mediated trans signalling. Any agent as describedherein may be used to inhibit IL-11-mediated cis and/or transsignalling.

In other examples, inhibition of IL-11 signalling may be achieved bydisrupting signalling pathways downstream of IL-11/IL-11Rα/gp130. Thatis, in some embodiments inhibition/antagonism of IL-11-mediatedsignalling comprises inhibition of a signalling pathway/process/factordownstream of signalling through the IL-11/IL-11 receptor complex.

In some embodiments inhibition/antagonism of IL-11-mediated signallingcomprises inhibition of signalling through an intracellular signallingpathway which is activated by the IL-11/IL-11 receptor complex. In someembodiments inhibition/antagonism of IL-11-mediated signalling comprisesinhibition of one or more factors whose expression/activity isupregulated as a consequence of signalling through the IL-11/IL-11receptor complex.

In some embodiments, the methods of the present invention employ agentscapable of inhibiting JAK/STAT signalling. In some embodiments, agentscapable of inhibiting JAK/STAT signalling are capable of inhibiting theaction of JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT4, STAT5A,STAT5B and/or STAT6. For example, agents may be capable of inhibitingactivation of JAK/STAT proteins, inhibiting interaction of JAK or STATproteins with cell surface receptors e.g. IL-11Rα or gp130, inhibitingphosphorylation of JAK proteins, inhibiting interaction between JAK andSTAT proteins, inhibiting phosphorylation of STAT proteins, inhibitingdimerization of STAT proteins, inhibiting translocation of STAT proteinsto the cell nucleus, inhibiting binding of STAT proteins to DNA, and/orpromoting degradation of JAK and/or STAT proteins. In some embodiments,a JAK/STAT inhibitor is selected from Ruxolitinib (Jakafi/Jakavi;Incyte), Tofacitinib (Xeljanz/Jakvinus; NIH/Pfizer), Oclacitinib(Apoquel), Baricitinib (Olumiant; Incyte/Eli Lilly), Filgotinib(G-146034/GLPG-0634; Galapagos Nev.), Gandotinib (LY-2784544; EliLilly), Lestaurtinib (CEP-701; Teva), Momelotinib (GS-0387/CYT-387;Gilead Sciences), Pacritinib (SB1518; CTI), PF-04965842 (Plizer),Upadacitinib (ABT-494; AbbVie), Peficitinib (ASP015K/JNJ-54781532;Astellas), Fedratinib (SAR302503; Celgene), Cucurbitacin I (JSI-124) andCHZ868.

In some embodiments, the methods of the present invention employ agentscapable of inhibiting MAPK/ERK signalling. In some embodiments, agentscapable of inhibiting MAPK/ERK signalling are capable of inhibiting theaction of GRB2, inhibiting the action of RAF kinase, inhibiting theaction of MEK proteins, inhibiting the activation of MAP3K/MAP2K/MAPKand/or Myc, and/or inhibiting the phosphorylation of STAT proteins. Insome embodiments, agents capable of inhibiting ERK signalling arecapable of inhibiting ERK p42/44. In some embodiments, an ERK inhibitoris selected from SCH772984, SC1, VX-Ile, DEL-22379, Sorafenib (Nexavar;Bayer/Onyx), SB590885, PLX4720, XL281, RAF265 (Novartis), encorafenib(LGX818/Braftovi; Array BioPharma), dabralenib (Tafinlar; GSK),vemurafenib (Zelboraf; Roche), cobimetinib (Cotellic; Roche), CI-1040,PD0325901, Binimetinib (MEK162/MEKTOVI; Array BioPharma), selumetinib(AZD6244; Array/AstraZeneca) and Trametinib (GSK1120212/Mekinist;Novartis). In some embodiments, the methods of the present inventionemploy agents capable of inhibiting c-Jun N-terminal kinase (JNK)signalling/activity. In some embodiments, agents capable of inhibitingJNK signalling/activity are capable of inhibiting the action and/orphosphorylation of a JNK (e.g. JNK1, JNK2). In some embodiments, a JNKinhibitor is selected from SP600125, CEP 1347. TCS JNK 6o, c-JUNpeptide, SU3327, AEG 3482, TCS JNK 5a, B178D3, IQ3, SR3576, IQ1S, JIP-1(153-163) and CC401 dihydrochloride.

Widjaja et al., bioRxiv (2019) 830018 demonstrates that NOX4 expressionand activity is upregulated by signalling through IL-11/IL-11Rα/gp130.NOX4 is an NADPH oxidase, and a source of reactive oxygen species (ROS).Expression of Nox4 is upregulated in transgenic mice withhepatocyte-specific Il11 expression, and primary human hepatocytesstimulated with IL11 upregulate NOX4 expression.

In some embodiments, the present invention employs agents capable ofinhibiting NOX4 expression (gene or protein expression) or function. Insome embodiments, the present invention employs agents capable ofinhibiting IL-11-mediated upregulation of NOX4 expression/function.Agents capable of inhibiting NOX4 expression or function may be referredto herein as NOX4 inhibitors. For example, a NOX4 inhibitor may becapable of reducing expression (e.g. gene and/or protein expression) ofNOX4, reducing the level of RNA encoding NOX4, reduce the level of NOX4protein, and/or reducing the level of a NOX4 activity (e.g. reducingNOX4-mediated NADPH oxidase activity and/or NOX4-mediated ROSproduction).

NOX4 inhibitors include a NOX4-binding molecules and molecules capableof reducing NOX4 expression. NOX4-binding inhibitors includepeptide/nucleic acid aptamers, antibodies (and antibody fragments) andfragments of interaction partners for NOX4 which behave as antagonistsof NOX4 function, and small molecules inhibitors of NOX4. Moleculescapable of reducing NOX4 expression include antisense RNA (e.g. siRNA,shRNA) to NOX4. In some embodiments, a NOX4 inhibitor is selected from aNOX4 inhibitor described in Altenhofer et al., Antioxid Redox Signal.(2015) 23(5): 406-427 or Augsburder et al., Redox Biol. (2019) 26:101272, such as GKT137831.

Binding Agents

In some embodiments, agents capable of inhibiting IL-11-mediatedsignalling may bind to IL-11. In some embodiments, agents capable ofinhibiting IL-11-mediated signalling may bind to a receptor for IL-11(e.g. IL-11Rα, gp130, or a complex containing IL-11Rα and/or gp130).Binding of such agents may inhibit IL-11-mediated signalling byreducing/preventing the ability of IL-11 to bind to receptors for IL-11,thereby inhibiting downstream signalling. Binding of such agents mayinhibit IL-11 mediated cis and/or trans-signalling byreducing/preventing the ability of IL-11 to bind to receptors for IL-11,e.g. IL-11Rα and/or gp130, thereby inhibiting downstream signalling.Agents may bind to trans-signalling complexes such as IL-11 and solubleIL-11Rα and inhibit gp130-mediated signalling.

Agents capable of binding to IL-11/an IL-11 containing complex or areceptor for IL-11 may be of any kind, but in some embodiments the agentmay be an antibody, an antigen-binding fragment thereof, a polypeptide,a peptide, a nucleic acid, an oligonucleotide, an aptamer or a smallmolecule. The agents may be provided in isolated or purified form, ormay be formulated as a pharmaceutical composition or medicament.

Antibodies and Antigen-Binding Fragments

In some embodiments, an agent capable of binding to IL-11/an IL-11containing complex or a receptor for IL-11 is an antibody, or anantigen-binding fragment thereof. In some embodiments, an agent capableof binding to IL-11/an IL-11 containing complex or a receptor for IL-11is a polypeptide, e.g. a decoy receptor molecule. In some embodiments,an agent capable of binding to IL-11/an IL-11 containing complex or areceptor for IL-11 may be an aptamer.

In some embodiments, an agent capable of binding to IL-11/an IL-11containing complex or a receptor for IL-11 is an antibody, or anantigen-binding fragment thereof. An “antibody” is used herein in thebroadest sense, and encompasses monoclonal antibodies, polyclonalantibodies, monospecific and multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, as long as they display binding tothe relevant target molecule.

In view of today's techniques in relation to monoclonal antibodytechnology, antibodies can be prepared to most antigens. Theantigen-binding portion may be a part of an antibody (for example a Fabfragment) or a synthetic antibody fragment (for example a single chainFv fragment [ScFv]). Monoclonal antibodies to selected antigens may beprepared by known techniques, for example those disclosed in “MonoclonalAntibodies: A manual of techniques”, H Zola (CRC Press, 1988) and in“Monoclonal Hybridoma Antibodies: Techniques and Applications”, J G RHurrell (CRC Press, 1982). Chimaeric antibodies are discussed byNeuberger et al (1988, 8th International Biotechnology Symposium Part 2,792-799). Monoclonal antibodies (mAbs) are particularly useful in themethods of the invention, and are a homogenous population of antibodiesspecifically targeting a single epitope on an antigen.

Polyclonal antibodies are also useful in the methods of the invention.Monospecilic polyclonal antibodies are preferred. Suitable polyclonalantibodies can be prepared using methods well known in the art.

Antigen-binding fragments of antibodies, such as Fab and Fab2 fragmentsmay also be used/provided as can genetically engineered antibodies andantibody fragments. The variable heavy (VH) and variable light (VL)domains of the antibody are involved in antigen recognition, a factfirst recognised by early protease digestion experiments. Furtherconfirmation was found by “humanisation” of rodent antibodies. Variabledomains of rodent origin may be fused to constant domains of humanorigin such that the resultant antibody retains the antigenicspecificity of the rodent parented antibody (Morrison et al (1984) Proc.Natl. Acad. Sd. USA 81, 6851-6855).

Antibodies and antigen-binding fragments according to the presentdisclosure comprise the complementarity-determining regions (CDRs) of anantibody which is capable of binding to the relevant target molecule(i.e. IL-11/an IL-11 containing complex/a receptor for IL-11).

Antibodies capable of binding to IL-11 include e.g. monoclonal mouseanti-human IL-11 antibody clone #22626; Catalog No. MAB218 (R&D Systems,MN, USA), used e.g. in Bockhorn et al. Nat. Commun. (2013) 4(0):1393,clone 6D9A (Abbiotec), clone KT8 (Abbiotec), clone M3103F11 (BioLegend),clone 1F1 (Abnova Corporation), clone 3C6 (Abnova Corporation), cloneGF1 (LifeSpan Biosciences), clone 13455 (Source BioScience), 11h3/19.6.1(Hermann et al., Arthritis Rheum. (1998) 41(8):1388-97), AB-218-NA (R&DSystems), X203 (Ng et al., Sci Transl Med. (2019) 11(511) pii: eaaw1237,which is also published as Ng, et al., “IL-11 is a therapeutic target inidiopathic pulmonary fibrosis.” bioRxiv 336537; doi:https://doi.org/10.1101/336537) and anti-IL-11 antibodies disclosed inUS 2009/0202533 A1, WO 99/59608 A2, WO 2018/109174 A2 and WO 2019/238882A1.

In particular, anti-IL-11 antibody clone 22626 (also known as MAB218)has been shown to be an antagonist of IL-11 mediated signalling, e.g. inSchaefer et al., Nature (2017) 552(7683):110-115. Monoclonal antibody11h3/19.6.1 is disclosed in Hermann et al., Arthritis Rheum. (1998)41(8):1388-97 to be a neutralising anti-IL-11 IgG1. AB-218-NA from R&DSystems, used e.g. in McCoy et al., BMC Cancer (2013) 13:16, is anotherexample of neutralizing anti-IL-11 antibody. WO 2018/109174 A2 and WO2019/238882 A1 disclose yet further exemplary anti-IL-11 antibodyantagonists of IL-11 mediated signalling. X203 (also referred to asEnx203) disclosed in Ng, et al., “IL-11 is a therapeutic target inidiopathic pulmonary fibrosis.” bioRxiv 336537; doi:https://doi.org/10.1101/336537 and WO 2019/238882 A1 is an anti-IL-11antibody antagonist of IL-11-mediated signalling, and comprises the VHregion according to SEQ ID NO:92 of WO 2019/238882 A1 (SEQ ID NO:22 ofthe present disclosure), and the VL region according to SEQ ID NO:94 ofWO 2019/238882 A1 (SEQ ID NO:23 of the present disclosure). Humanisedversions of the X203 are described in WO 2019/238882 A1, includinghEnx203 which comprises the VH region according to SEQ ID NO: 17 of WO2019/238882 A1 (SEQ ID NO:30 of the present disclosure), and the VLregion according to SEQ ID NO:122 of WO 2019/238882 A1 (SEQ ID NO:31 ofthe present disclosure). Enx108A is a further example of an anti-IL-11antibody antagonist of IL-11-mediated signalling, and comprises the VHregion according to SEQ ID NO:8 of WO 2019/238882 A1 (SEQ ID NO:26 ofthe present disclosure), and the VL region according to SEQ ID NO:20 ofWO 2019/238882 A1 (SEQ ID NO:27 of the present disclosure).

Antibodies capable of binding to IL-11Rα include e.g. monoclonalantibody clone 025 (Sino Biological), clone EPR5446 (Abcam), clone473143 (R & D Systems), clones 8E2, 8D10 and 8E4 and theaffinity-matured variants of 8E2 described in US 2014/0219919 A1, themonoclonal antibodies described in Blanc et al (J. Immunol Methods. 2000Jul. 31; 241(1-2); 43-59), X209 (Widjaja et al., Gastroenterology (2019)157(3):777-792, which is also published as Widjaja, et al., “IL-11neutralising therapies target hepatic stellate cell-induced liverinflammation and fibrosis in NASH.” bioRxiv 470062; doi:https://doi.org/10.1101/470062) antibodies disclosed in WO 2014121325 A1and US 2013/0302277 A1, and anti-IL-11Rα antibodies disclosed in US2009/0202533 A1, WO 99/59608 A2, WO 2018/109170 A2 and WO 2019/238884A1.

In particular, anti-IL-11Rα antibody clone 473143 (also known asMAB1977) has been shown to be an antagonist of IL-11 mediatedsignalling, e.g. in Schaefer et al., Nature (2017) 552(7683):110-115. US2014/0219919 A1 provides sequences for anti-human IL-11Rα antibodyclones 8E2, 8D10 and 8E4, and discloses their ability to antagoniseIL-11 mediated signalling—see e.g. [0489] to [0490] of US 2014/0219919A1. US 2014/0219919 A1 moreover provides sequence information for anadditional 62 affinity-matured variants of clone 8E2, 61 of which aredisclosed to antagonise IL-11 mediated signalling—see Table 3 of US2014/0219919 A1. WO 2018/109170 A2 and WO 2019/238884 A1 disclose yetfurther exemplary anti-IL-11Rα antibody antagonists of IL-11 mediatedsignalling. X209 (also referred to as Enx209) disclosed in Widjaja, etal., “IL-11 neutralising therapies target hepatic stellate cell-inducedliver inflammation and fibrosis in NASH.” bioRxiv 470062; doi:https://doi.org/i0.1101/470062 and WO 2019/238884 A1 is an anti-IL-11Rαantibody antagonist of IL-11-mediated signalling, and comprises the VHregion according to SEQ ID NO:7 of WO 2019/238884 A1 (SEQ ID NO:24 ofthe present disclosure), and the VL region according to SEQ ID NO:14 ofWO 2019/238884 A1 (SEQ ID NO:25 of the present disclosure). Humanisedversions of the X209 are described in WO 2019/238884 A1, includinghEnx209 which comprises the VH region according to SEQ ID NO: 1 of WO2019/238884 A1 (SEQ ID NO:32 of the present disclosure), and the VLregion according to SEQ ID NO:17 of WO 2019/238884 A1 (SEQ ID NO:33 ofthe present disclosure).

The skilled person is well aware of techniques for producing antibodiessuitable for therapeutic use in a given species/subject. For example,procedures for producing antibodies suitable for therapeutic use inhumans are described in Park and Smolen Advances in Protein Chemistry(2001) 56: 369-421 (hereby incorporated by reference in its entirely).

Antibodies to a given target protein (e.g. IL-11 or IL-11Rα) can beraised in model species (e.g. rodents, lagomorphs), and subsequentlyengineered in order to improve their suitability for therapeutic use ina given species/subject. For example, one or more amino acids ofmonoclonal antibodies raised by immunisation of model species can besubstituted to arrive at an antibody sequence which is more similar tohuman germline immunoglobulin sequences (thereby reducing the potentialfor anti-xenogenic antibody immune responses in the human subjecttreated with the antibody). Modifications in the antibody variabledomains may focus on the framework regions in order to preserve theantibody paratope. Antibody humanisation is a matter of routine practicein the art of antibody technology, and is reviewed e.g. in Almagro andFransson, Frontiers in Bioscience (2008) 13:1619-1633, Safdari et al.,Biotechnology and Genetic Engineering Reviews (2013) 29(2): 175-186 andLo et al., Microbiology Spectrum (2014) 2(1), all of which are herebyincorporated by reference in their entirety. The requirement forhumanisation can be circumvented by raising antibodies to a given targetprotein (e.g. IL-11 or IL-11Rα) in transgenic model species expressinghuman immunoglobulin genes, such that the antibodies raised in suchanimals are fully-human (described e.g. in Bruggemann et al., ArchImmunol Ther Exp (Warsz) (2015) 63(2):101-108, which is herebyincorporated by reference in its entirety).

Phage display techniques may also be employed to the identification ofantibodies to a given target protein (e.g. IL-11 or IL-11Rα), and arewell known to the skilled person. The use of phage display for theidentification of fully human antibodies to human target proteins isreviewed e.g. in Hoogenboom, Nat. Biotechnol. (2005) 23, 1105-1116 andChan et al., International Immunology (2014) 26(12): 649-657, which arehereby incorporated by reference in their entirety.

The antibodies/fragments may be antagonist antibodies/fragments thatinhibit or reduce a biological activity of IL-11. Theantibodies/fragments may be neutralising antibodies that neutralise thebiological effect of IL-11, e.g. its ability to stimulate productivesignalling via an IL-11 receptor. Neutralising activity may be measuredby ability to neutralise IL-11 induced proliferation in the T11 mouseplasmacytoma cell line (Nordan, R. P. et al. (1987) J. Immunol.139:813).

IL-11- or IL-11Rα-binding antibodies can be evaluated for the ability toantagonise IL-11-mediated signalling, e.g. using the assay described inUS 2014/0219919 A1 or Blanc et al (J. Immunol Methods. 2000 Jul. 31;241(1-2); 43-59. Briefly, IL-11- and IL-11Rα-binding antibodies can beevaluated in vitro for the ability to inhibit proliferation of Ba/F3cells expressing IL-11Rα and gp130 from the appropriate species, inresponse to stimulation with IL-11 from the appropriate species.Alternatively, IL-11- and IL-11Rα-binding antibodies can be analysed invitro for the ability to inhibit the fibroblast-to-myofibroblasttransition following stimulation of fibroblasts with TGFβ1, byevaluation of αSMA expression (as described e.g. in WO 2018/109174 A2(Example 6) and WO 2018/109170 A2 (Example 6), Ng et al., Sci TranslMed. (2019) 11(511) pii: eaaw1237 and Widjaja et al., Gastroenterology(2019) 157(3):777-792).

Antibodies generally comprise six CDRs; three in the light chainvariable region (VL): LC-CDR1, LC-CDR2, LC-CDR3, and three in the heavychain variable region (VH): HC-CDR1, HC-CDR2 and HC-CDR3. The six CDRstogether define the paratope of the antibody, which is the part of theantibody which binds to the target molecule. The VH region and VL regioncomprise framework regions (FRs) either side of each CDR, which providea scaffold for the CDRs. From N-terminus to C-terminus, VH regionscomprise the following structure: Nterm-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-Cterm; and VL regions comprise the following structure: Nterm-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-Cterm.

There are several different conventions for defining antibody CDRs andFRs, such as those described in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991), Chothia et al., J. Mol.Biol. 196:901-917 (1987), and VBASE2, as described in Retter et al.,Nucl. Acids Res. (2005) 33 (suppl 1): D671-D674. The CDRs and FRs of theVH regions and VL regions of the antibodies described herein are definedaccording to the Kabat system.

In some embodiments an antibody, or an antigen-binding fragment thereof,according to the present disclosure is derived from an antibody whichbinds specifically to IL-11 (e.g. Enx108A, Enx203 or hEnx203). In someembodiments an antibody, or an antigen-binding fragment thereof,according to the present disclosure is derived from an antibody whichbinds specifically to IL-11Rα (e.g. Enx209 or hEnx209).

Antibodies and antigen-binding fragments according to the presentdisclosure preferably inhibit IL-11-mediated signalling. Suchantibodies/antigen-binding fragments may be described as beingantagonists of IL-11-mediated signalling, and/or may be described ashaving the ability to neutralise IL-11-mediated signalling.

In some embodiments, the antibody/antigen-binding fragment comprises theCDRs of an antibody which binds to IL-11. In some embodiments theantibody/antigen-binding fragment comprises the CDRs of, or CDRs derivedfrom, the CDRs of an IL-11-binding antibody described herein (e.g.Enx108A, Enx203 or hEnx203).

In some embodiments the antibody/antigen-binding fragment comprises a VHregion incorporating the following CDRs:

(1)

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:34    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:35    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:36,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

In some embodiments the antibody/antigen-binding fragment comprises a VLregion incorporating the following CDRs:

(2)

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:37    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:38    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:39,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1. LC-CDR2, or LC-CDR3 are substituted with        another amino acid.

In some embodiments the antibody/antigen-binding fragment comprises a VHregion incorporating the following CDRs:

(3)

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:40    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:41    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:42.    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

In some embodiments the antibody/antigen-binding fragment comprises a VLregion incorporating the following CDRs:

(4)

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:43    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:44    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:45,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1. LC-CDR2, or LC-CDR3 are substituted with        another amino acid.

In some embodiments the antibody/antigen-binding fragment comprises a VHregion incorporating the CDRs according to (1), and a VL regionincorporating the CDRs according to (2). In some embodiments theantibody/antigen-binding fragment comprises a VH region incorporatingthe CDRs according to (3), and a VL region incorporating the CDRsaccording to (4).

In some embodiments the antibody/antigen-binding fragment comprises theVH region and the VL region of an antibody which binds to IL-11. In someembodiments the antibody/antigen-binding fragment comprises the VHregion and VL region of, or a VH region and VL region derived from, theVH region and VL region of an IL-11-binding antibody described herein(e.g. Enx108A, Enx203 or hEnx203).

In some embodiments the antibody/antigen-binding fragment comprises a VHregion comprising an amino acid sequence having at least 70% sequenceidentity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequenceidentity to the amino acid sequence of SEQ ID NO:26. In some embodimentsthe antibody/antigen-binding fragment comprises a VL region comprisingan amino acid sequence having at least 70% sequence identity morepreferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity tothe amino acid sequence of SEQ ID NO:27. In some embodiments theantibody/antigen-binding fragment comprises a VH region comprising anamino acid sequence having at least 70% sequence identity morepreferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity tothe amino acid sequence of SEQ ID NO:26 and a VL region comprising anamino acid sequence having at least 70% sequence identity morepreferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity tothe amino acid sequence of SEQ ID NO:27.

In some embodiments the antibody/antigen-binding fragment comprises a VHregion comprising an amino acid sequence having at least 70% sequenceidentity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97/c, 98%, 99%, or 100%,sequence identity to the amino acid sequence of SEQ ID NO:22. In someembodiments the antibody/antigen-binding fragment comprises a VL regioncomprising an amino acid sequence having at least 70% sequence identitymore preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identityto the amino acid sequence of SEQ ID NO:23. In some embodiments theantibody/antigen-binding fragment comprises a VH region comprising anamino acid sequence having at least 70% sequence identity morepreferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity tothe amino acid sequence of SEQ ID NO:22 and a VL region comprising anamino acid sequence having at least 70% sequence identity morepreferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity tothe amino acid sequence of SEQ ID NO:23.

In some embodiments the antibody/antigen-binding fragment comprises a VHregion comprising an amino acid sequence having at least 70% sequenceidentity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequenceidentity to the amino acid sequence of SEQ ID NO:30. In some embodimentsthe antibody/antigen-binding fragment comprises a VL region comprisingan amino acid sequence having at least 70% sequence identity morepreferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1009%, sequence identity tothe amino acid sequence of SEQ ID NO:31. In some embodiments theantibody/antigen-binding fragment comprises a VH region comprising anamino acid sequence having at least 70% sequence identity morepreferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity tothe amino acid sequence of SEQ ID NO:30 and a VL region comprising anamino acid sequence having at least 70% sequence identity morepreferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity tothe amino acid sequence of SEQ ID NO:31.

In some embodiments, the antibody/antigen-binding fragment comprises theCDRs of an antibody which binds to IL-11Rα. In some embodiments theantibody/antigen-binding fragment comprises the CDRs of, or CDRs derivedfrom, the CDRs of an IL-11Rα-binding antibody described herein (e.g.Enx209 or hEnx209).

In some embodiments the antibody/antigen-binding fragment comprises a VHregion incorporating the following CDRs:

(5)

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:46    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:47    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

In some embodiments the antibody/antigen-binding fragment comprises a VLregion incorporating the following CDRs:

(6)

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:49    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:50    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:51,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with        another amino acid.

In some embodiments the antibody/antigen-binding fragment comprises a VHregion incorporating the CDRs according to (5), and a VL regionincorporating the CDRs according to (6).

In some embodiments the antibody/antigen-binding fragment comprises theVH region and the VL region of an antibody which binds to IL-11Rα. Insome embodiments the antibody/antigen-binding fragment comprises the VHregion and VL region of, or a VH region and VL region derived from, theVH region and VL region of an IL-11Rα-binding antibody described herein(e.g. Enx209 or hEnx209).

In embodiments in accordance with the present invention in which one ormore amino acids of a reference amino acid sequence (e.g. a CDRsequence, VH region sequence or VL region sequence described herein) aresubstituted with another amino acid, the substitutions may conservativesubstitutions, for example according to the following Table. In someembodiments, amino acids in the same block in the middle column aresubstituted. In some embodiments, amino acids in the same line in therightmost column are substituted:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

In some embodiments, substitution(s) may be functionally conservative.That is, in some embodiments the substitution may not affect (or may notsubstantially affect) one or more functional properties (e.g. targetbinding) of the antibody/fragment comprising the substitution relativeto the equivalent unsubstituted molecule.

In some embodiments, substitution(s) relative to a reference VH regionor VL region sequence may be focused in a particular region or regionsof the VH region or VL region sequence. For example, variation from areference VH region or VL region sequence may be focused in one or moreof the framework regions (FR1, FR2, FR3 and/or FR4).

Antibodies and antigen-binding fragments according to the presentdisclosure may be designed and prepared using the sequences ofmonoclonal antibodies (mAbs) capable of binding to the relevant targetmolecule. Antigen-binding regions of antibodies, such as single chainvariable fragment (scFv), Fab and Fab2 fragments may also beused/provided. An ‘antigen-binding region’ or ‘antigen binding fragment’is any fragment of an antibody which is capable of binding to the targetfor which the given antibody is specific.

In some embodiments the antibodies/fragments comprise the VL and VHregions of an antibody which is capable of binding to IL-11, an IL-11containing complex, or a receptor for IL-11. The VL and VH region of anantigen-binding region of an antibody together constitute the Fv region.In some embodiments the antibodies/fragments comprise or consist of theFv region of an antibody which is capable of binding to IL-11, an IL-11containing complex, or a receptor for IL-11. The Fv region may beexpressed as a single chain wherein the VH and VL regions are covalentlylinked, e.g. by a flexible oligopeptide. Accordingly,antibodies/fragments may comprise or consist of an scFv comprising theVL and VH regions of an antibody which is capable of binding to IL-11,an IL-11 containing complex, or a receptor for IL-11.

The VL and light chain constant (CL) region, and the VH region and heavychain constant 1 (CH1) region of an antigen-binding region of anantibody together constitute the Fab region. In some embodiments theantibodies/fragments comprise or consist of the Fab region of anantibody which is capable of binding to IL-11, an IL-11 containingcomplex, or a receptor for IL-11.

In some embodiments, antibodies/fragments comprise, or consist of, wholeantibody capable of binding to IL-11, an IL-11 containing complex, or areceptor for IL-11. A “whole antibody” refers to an antibody having astructure which is substantially similar to the structure of animmunoglobulin (Ig). Different kinds of immunoglobulins and theirstructures are described e.g. in Schroeder and Cavacini J Allergy ClinImmunol. (2010) 125(202): S41-S52, which is hereby incorporated byreference in its entirety. Immunoglobulins of type G (i.e. IgG) are ˜150kDa glycoproteins comprising two heavy chains and two light chains. FromN- to C-terminus, the heavy chains comprise a VH followed by a heavychain constant region comprising three constant domains (CH1, CH2, andCH3), and similarly the light chain comprises a VL followed by a CL.Depending on the heavy chain, immunoglobulins may be classed as IgG(e.g. IgG1, IgG2, IgG3, IgG4), IgA (e.g. IgA1, IgA2), IgD, IgE, or IgM.The light chain may be kappa (κ) or lambda (λ).

In some embodiments the antibody/antigen-binding fragment of the presentdisclosure comprises an immunoglobulin heavy chain constant sequence. Insome embodiments, an immunoglobulin heavy chain constant sequence may bea human immunoglobulin heavy chain constant sequence. In someembodiments the immunoglobulin heavy chain constant sequence is, or isderived from, the heavy chain constant sequence of an IgG (e.g. IgG1,IgG2, IgG3, IgG4), IgA (e.g. IgA1, IgA2), IgD, IgE or IgM, e.g. a humanIgG (e.g. hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g. hIgA1, hIgA2), hIgD,hIgE or hIgM. In some the immunoglobulin heavy chain constant sequenceis, or is derived from, the heavy chain constant sequence of a humanIgG1 allotype (e.g. G1m1, G1m2, G1m3 or G1m17).

In some embodiments the immunoglobulin heavy chain constant sequence is,or is derived from, the constant region sequence of human immunoglobulinG 1 constant (IGHG1; UniProt: P01857-1, v1). In some embodiments theimmunoglobulin heavy chain constant sequence is, or is derived from, theconstant region sequence of human immunoglobulin G 1 constant (IGHG1:UniProt: P01857-1, v1) comprising substitutions K214R, D356E and L358M(i.e. the G1m3 allotype). In some embodiments theantibody/antigen-binding fragment comprises an amino acid sequencehaving at least 70% sequence identity more preferably one of at least75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence ofSEQ ID NO:52.

In some embodiments the immunoglobulin heavy chain constant sequence is,or is derived from, the constant region sequence of human immunoglobulinG 4 constant (IGHG4; UniProt: P01861, v1). In some embodiments theimmunoglobulin heavy chain constant sequence is, or is derived from, theconstant region sequence of human immunoglobulin G 4 constant (IGHG4;UniProt: P01861, v1) comprising substitutions S241P and/or L248E. TheS241P mutation is hinge stabilising while the L248E mutation furtherreduces the already low ADCC effector function of IgG4 (Davies andSutton, Immunol Rev. 2015 November; 268(1):139-159; Angal et al MolImmunol. 1993 January; 30(1):105-8). In some embodiments theantibody/antigen-binding fragment comprises an amino acid sequencehaving at least 70% sequence identity more preferably one of at least75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence ofSEQ ID NO:53.

In some embodiments the antibody/antigen-binding fragment of the presentdisclosure comprises an immunoglobulin light chain constant sequence. Insome embodiments, an immunoglobulin light chain constant sequence may bea human immunoglobulin light chain constant sequence. In someembodiments the immunoglobulin light chain constant sequence is, or isderived from, a kappa (κ) or lambda (λ) light chain, e.g. humanimmunoglobulin kappa constant (IGKC; Cκ; UniProt: P01834-1, v2), orhuman immunoglobulin lambda constant (IGLC; Cλ), e.g. IGLC1 (UniProt:P0CG04-1, v1), IGLC2 (UniProt: P0DOY2-1, v1), IGLC3 (UniProt: P0DOY3-1,v1), IGLC6 (UniProt: P0CF74-1, v1) or IGLC7 (UniProt: A0M8Q6-1, v3).

In some embodiments the antibody/antigen-binding fragment comprises animmunoglobulin light chain constant sequence. In some embodiments theimmunoglobulin light chain constant sequence is, or is derived fromhuman immunoglobulin kappa constant (IGKC; Cκ; UniProt: P01834-1, v2;SEQ ID NO:90). In some embodiments the immunoglobulin light chainconstant sequence is a human immunoglobulin lambda constant (IGLC; Cλ),e.g. IGLC1, IGLC2, IGLC3, IGLC6 or IGLC7. In some embodiments theantibody/antigen-binding fragment comprises an amino acid sequencehaving at least 70% sequence identity more preferably one of at least75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence ofSEQ ID NO:54. In some embodiments the antibody/antigen-binding fragmentcomprises an amino acid sequence having at least 70% sequence identitymore preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identityto the amino acid sequence of SEQ ID NO:55.

In some embodiments, the antibody/antigen-binding fragment comprises:(i) a polypeptide comprising or consisting of an amino acid sequencehaving at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityto the amino acid sequence of SEQ ID NO:28, and (ii) a polypeptidecomprising or consisting of an amino acid sequence having at least 70%,preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% amino acid sequence identity to the amino acid sequenceof SEQ ID NO-29.

In some embodiments, the antibody/antigen-binding fragment comprises:(i) a polypeptide comprising or consisting of an amino acid sequencehaving at least 70%, preferably one of 75%, 80%, 85%, 90%₆, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityto the amino acid sequence of SEQ ID NO:56, and (ii) a polypeptidecomprising or consisting of an amino acid sequence having at least 70%,preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% amino acid sequence identity to the amino acid sequenceof SEQ ID NO:57.

In some embodiments, the antibody/antigen-binding fragment comprises:(i) a polypeptide comprising or consisting of an amino acid sequencehaving at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityto the amino acid sequence of SEQ ID NO:58, and (ii) a polypeptidecomprising or consisting of an amino acid sequence having at least 70%,preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% amino acid sequence identity to the amino acid sequenceof SEQ ID NO:59.

Fab, Fv, ScFv and dAb antibody fragments can all be expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of the said fragments.

Whole antibodies, and F(ab′)2 fragments are “bivalent”. By “bivalent” wemean that the said antibodies and F(ab′)2 fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv and dAb fragments aremonovalent, having only one antigen combining site. Synthetic antibodiescapable of binding to IL-11, an IL-11 containing complex, or a receptorfor IL-11 may also be made using phage display technology as is wellknown in the art.

Antibodies may be produced by a process of affinity maturation in whicha modified antibody is generated that has an improvement in the affinityof the antibody for antigen, compared to an unmodified parent antibody.Affinity-matured antibodies may be produced by procedures known in theart, e.g., Marks et al., Rio/Technology 10:779-783 (1992): Barbas et al.Proc Nat. Acad. Sci. USA 91:3809-3813 (1994): Schier et al. Gene169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995);Jackson et al., J. Immunol. 154(7):331 0-15 9 (1995); and Hawkins el al,J. Mol. Biol. 226:889-896 (1992).

Antibodies/fragments include bi-specific antibodies, e.g. composed oftwo different fragments of two different antibodies, such that thebi-specific antibody binds two types of antigen. The bispecific antibodycomprises an antibody/fragment as described herein capable of binding toIL-11, an IL-11 containing complex, or a receptor for IL-11. Theantibody may contain a different fragment having affinity for a secondantigen, which may be any desired antigen. Techniques for thepreparation of bi-specific antibodies are well known in the art, e.g.see Mueller, D et al., (2010 Biodrugs 24 (2): 89-98), Wozniak-Knopp G etal., (2010 Protein Eng Des 23 (4): 289-297), and Baeuerle, P A et al.,(2009 Cancer Res 69 (12): 4941-4944). Bispecific antibodies andbispecific antigen-binding fragments may be provided in any suitableformat, such as those formats described in Kontermann MAbs 2012, 4(2):182-197, which is hereby incorporated by reference in its entirety. Forexample, a bispecific antibody or bispecific antigen-binding fragmentmay be a bispecific antibody conjugate (e.g. an IgG2, F(ab′)2 orCovX-Body), a bispecific IgG or IgG-like molecule (e.g. an IgG,scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, 2 in 1-IgG,mAb2, or Tandemab common LC), an asymmetric bispecific IgG or IgG-likemolecule (e.g. a kih IgG, kih IgG common LC, CrossMab, kih IgG-scFab,mAb-Fv, charge pair or SEED-body), a small bispecific antibody molecule(e.g. a Diabody (db), dsDb, DART, scDb, tandAbs, tandem scFv (taFv),tandem dAbNHH, triple body, triple head, Fab-scFv, or F(ab′)2-scFv2), abispecific Fc and CH3 fusion protein (e.g. a taFv-Fc, Di-diabody,scDb-CH3, scFv-Fc-scFv. HCAb-VHH, scFv-kih-Fc, or scFv-kih-CH3), or abispecific fusion protein (e.g. a scFv2-albumin, scDb-albumin,taFv-toxin, DNL-Fab3, DNL-Fab4-IgG, DNL-Fab4-IgG-cytokine2). See inparticular FIG. 2 of Kontermann MAbs 2012, 4(2): 182-19.

Methods for producing bispecific antibodies include chemicallycrosslinking antibodies or antibody fragments, e.g. with reducibledisulphide or non-reducible thioether bonds, for example as described inSegal and Bast, 2001. Production of Bispecific Antibodies. CurrentProtocols in Immunology. 14:IV:2.13:2.13.1-2.13.16, which is herebyincorporated by reference in its entirety. For example,N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used tochemically crosslink e.g. Fab fragments via hinge region SH— groups, tocreate disulfide-linked bispecific F(ab)2 heterodimers.

Other methods for producing bispecific antibodies include fusingantibody-producing hybridomas e.g. with polyethylene glycol, to producea quadroma cell capable of secreting bispecific antibody, for example asdescribed in D. M. and Bast, B. J. 2001. Production of BispecificAntibodies. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16.

Bispecific antibodies and bispecific antigen-binding fragments can alsobe produced recombinantly, by expression from e.g. a nucleic acidconstruct encoding polypeptides for the antigen binding molecules, forexample as described in Antibody Engineering: Methods and Protocols,Second Edition (Humana Press. 2012), at Chapter 40: Production ofBispecific Antibodies: Diabodies and Tandem scFv (Hornig andFArber-Schwarz), or French, How to make bispecific antibodies. MethodsMol. Med. 2000; 40:333-339.

For example, a DNA construct encoding the light and heavy chain variabledomains for the two antigen binding domains (i.e. the light and heavychain variable domains for the antigen binding domain capable of bindingto IL-11, an IL-11 containing complex, or a receptor for IL-11, and thelight and heavy chain variable domains for the antigen binding domaincapable of binding to another target protein), and including sequencesencoding a suitable linker or dimerization domain between the antigenbinding domains can be prepared by molecular cloning techniques.Recombinant bispecific antibody can thereafter be produced by expression(e.g. in vitro) of the construct in a suitable host cell (e.g. amammalian host cell), and expressed recombinant bispecific antibody canthen optionally be purified.

Decoy Receptors

Peptide or polypeptide based agents capable of binding to IL-11 or IL-11containing complexes may be based on the IL-11 receptor, e.g. an IL-11binding fragment of an IL-11 receptor.

In some embodiments, the binding agent may comprise an IL-11-bindingfragment of the IL-11Rα chain, and may preferably be soluble and/orexclude one or more, or all, of the transmembrane domain(s). In someembodiments, the binding agent may comprise an IL-11-binding fragment ofgp130, and may preferably be soluble and/or exclude one or more, or all,of the transmembrane domain(s). Such molecules may be described as decoyreceptors. Binding of such agents may inhibit IL-11 mediated cis and/ortrans-signalling by reducing/preventing the ability of IL-11 to bind toreceptors for IL-11, e.g. IL-11Rα or gp130, thereby inhibitingdownstream signalling.

Curtis et al (Blood 1997 Dec. 1; 90 (11):4403-12) report that a solublemurine IL-11 receptor alpha chain (sIL-11R) was capable of antagonizingthe activity of IL-11 when tested on cells expressing the transmembraneIL-11R and gp130. They proposed that the observed IL-11 antagonism bythe sIL-11R depends on limiting numbers of gp130 molecules on cellsalready expressing the transmembrane IL-11R.

The use of soluble decoy receptors as the basis for inhibition of signaltransduction and therapeutic intervention has also been reported forother signalling molecule:receptor pairs, e.g. VEGF and the VEGFreceptor (De-Chao Yu et al., Molecular Therapy (2012); 20 5, 938-947;Konner and Dupont Clin Colorectal Cancer 2004 October; 4 Suppl 2:S81-5).

As such, in some embodiments a binding agent may be a decoy receptor,e.g. a soluble receptor for IL-11 and/or IL-11 containing complexes.Competition for IL-11 and/or IL-11 containing complexes provided by adecoy receptor has been reported to lead to IL-11 antagonist action(Curtis et al., supra). Decoy IL-11 receptors are also described in WO2017/103108 A1 and WO 2018/109168 A1, which are hereby incorporated byreference in their entirety.

Decoy IL-11 receptors preferably bind IL-11 and/or IL-11 containingcomplexes, and thereby make these species unavailable for binding togp130, IL-11Rα and/or gp130:IL-11Rα receptors. As such, they act as‘decoy’ receptors for IL-11 and IL-11 containing complexes, much in thesame way that etanercept acts as a decoy receptor for TNFα.IL-11-mediated signalling is reduced as compared to the level ofsignalling in the absence of the decoy receptor.

Decoy IL-11 receptors preferably bind to IL-11 through one or morecytokine binding modules (CBMs). The CBMs are, or are derived from orhomologous to, the CBMs of naturally occurring receptor molecules forIL-11. For example, decoy IL-11 receptors may comprise, or consist of,one or more CBMs which are from, are derived from or homologous to theCBM of gp130 and/or IL-11Rα.

In some embodiments, a decoy IL-11 receptor may comprise, or consist of,an amino acid sequence corresponding to the cytokine binding module ofgp130. In some embodiments, a decoy IL-11 receptor may comprise an aminoacid sequence corresponding to the cytokine binding module of IL-11Rα.Herein, an amino acid sequence which ‘corresponds’ to a reference regionor sequence of a given peptide/polypeptide has at least 60%, e.g. one ofat least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity to the amino acid sequence of thereference region/sequence.

In some embodiments a decoy receptor may be able to bind IL-11, e.g.with binding affinity of at least 100 μM or less, optionally one of 10μM or less, 1 μM or less, 100 nM or less, or about 1 to 100 nM. In someembodiments a decoy receptor may comprise all or part of the IL-11binding domain and may optionally lack all or part of the transmembranedomains. The decoy receptor may optionally be fused to an immunoglobulinconstant region, e.g. IgG Fc region.

Inhibitors

The present invention contemplates the use of inhibitor moleculescapable of binding to one or more of IL-11, an IL-11 containing complex,IL-11Rα, gp130, or a complex containing IL-11Rα and/or gp130, andinhibiting IL-11 mediated signalling.

In some embodiments the agent is a peptide- or polypeptide-based bindingagent based on IL-11, e.g. mutant, variant or binding fragment of IL-11.Suitable peptide or polypeptide based agents may bind to a receptor forIL-11 (e.g. IL-11Rα, gp130, or a complex containing IL-11Rα and/orgp130) in a manner that does not lead to initiation of signaltransduction, or which produces sub-optimal signalling. IL-11 mutants ofthis kind may act as competitive inhibitors of endogenous IL-11.

For example, W147A is an IL-11 antagonist in which the amino acid 147 ismutated from a tryptophan to an alanine, which destroys the so-called‘site III’ of IL-11. This mutant can bind to IL-11Rα, but engagement ofthe gp130 homodimer fails, resulting in efficient blockade of IL-11signalling (Underhill-Day et al., 2003; Endocrinology 2003 August;144(8):3406-14). Lee et al (Am J respire Cell Mol Biol. 2008 December;39(6):739-746) also report the generation of an IL-11 antagonist mutant(a “mutein”) capable of specifically inhibiting the binding of IL-11 toIL-11Rα. IL-11 muteins are also described in WO 2009/052588 A1.

Menkhorst et al (Biology of Reproduction May 1, 2009 vol. 80 no. 5920-927) describe a PEGylated IL-11 antagonist, PEGIL11A (CSL Limited.Parkvill, Victoria, Australia) which is effective to inhibit IL-11action in female mice.

Pasqualini et al. Cancer (2015) 121(14):2411-2421 describe aligand-directed, peptidomimetic drug, bone metastasis-targetingpeptidomimetic-11 (BMTP-11) capable of binding to IL-11Rα.

In some embodiments a binding agent capable of binding to a receptor forIL-11 may be provided in the form of a small molecule inhibitor of oneof IL-11R, gp130, or a complex containing IL-11Rα and/or gp130. In someembodiments a binding agent may be provided in the form of a smallmolecule inhibitor of IL-11 or an IL-11 containing complex, e.g. IL-11inhibitor described in Lay et al., Int. J. Oncol. (2012); 41(2):759-764, which is hereby incorporated by reference in its entirety.

Aptamers

In some embodiments, an agent capable of binding to IL-11/an IL-11containing complex or a receptor for IL-11 (e.g. IL-11Rα, gp130, or acomplex containing IL-11Rα and/or gp130) is an aptamer. Aptamers, alsocalled nucleic acid/peptide ligands, are nucleic acid or peptidemolecules characterised by the ability to bind to a target molecule withhigh specificity and high affinity. Almost every aptamer identified todate is a non-naturally occurring molecule.

Aptamers to a given target (e.g. IL-11, an IL-11 containing complex or areceptor for IL-11) may be identified and/or produced by the method ofSystematic Evolution of Ligands by EXponential enrichment (SELEX™), orby developing SOMAmers (slow off-rate modified aptamers) (Gold L et al.(2010) PLoS ONE 5(12):e15004). Aptamers and SELEX are described in Tuerkand Gold, Science (1990) 249(4968):505-10, and in WO 91/19813. Applyingthe SELEX and the SOMAmer technology includes for instance addingfunctional groups that mimic amino acid side chains to expand theaptamer's chemical diversity. As a result high affinity aptamers for atarget may be enriched and identified.

Aptamers may be DNA or RNA molecules and may be single stranded ordouble stranded. The aptamer may comprise chemically modified nucleicacids, for example in which the sugar and/or phosphate and/or base ischemically modified. Such modifications may improve the stability of theaptamer or make the aptamer more resistant to degradation and mayinclude modification at the 2′ position of ribose.

Aptamers may be synthesised by methods which are well known to theskilled person. For example, aptamers may be chemically synthesised,e.g. on a solid support. Solid phase synthesis may use phosphoramiditechemistry. Briefly, a solid supported nucleotide is detritylated, thencoupled with a suitably activated nucleoside phosphoramidite to form aphosphite triester linkage. Capping may then occur, followed byoxidation of the phosphite triester with an oxidant, typically iodine.The cycle may then be repeated to assemble the aptamer (e.g., see Sinha,N. D.; Biernat, J.; McManus, J.; Köster, H. Nucleic Acids Res. 1984, 12,4539; and Beaucage, S. L.; Lyer, R. P. (1992). Tetrahedron 48 (12):2223).

Suitable nucleic acid aptamers may optionally have a minimum length ofone of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40nucleotides. Suitable nucleic acid aptamers may optionally have amaximum length of one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides.Suitable nucleic acid aptamers may optionally have a length of one of10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80nucleotides.

Aptamers may be peptides selected or engineered to bind specific targetmolecules. Peptide aptamers and methods for their generation andidentification are reviewed in Reverdatto et al., Curr Top Med Chem.(2015) 15(12):1082-101, which is hereby incorporated by reference in itsentirety. Peptide aptamers may optionally have a minimum length of oneof 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. Peptide aptamers mayoptionally have a maximum length of one of 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids. Suitablepeptide aptamers may optionally have a length of one of 2-30, 2-25,2-20, 5-30, 5-25 or 5-20 amino acids.

Aptamers may have K_(D)'s in the nM or pM range, e.g. less than one of500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 μM, 100 μM.

Properties of IL-11 Binding Agents

Agents capable of binding to IL-11/an IL-11 containing complex or areceptor for IL-11 according to the present invention may exhibit one ormore of the following properties:

-   -   Specific binding to IL-11/IL-11 containing complex or a receptor        for IL-11;    -   Binding to IL-11/IL-11 containing complex, or a receptor for        IL-11, with a KD of 10 μM or less, preferably one of ≤5 μM ≤1        μM, ≤500 nM, ≤100 nM, ≤10 nM, ≤1 nM or ≤100 pM;    -   Inhibition of interaction between IL-11 and IL-11Rα;    -   Inhibition of interaction between IL-11 and gp130;    -   Inhibition of interaction between IL-11 and IL-11Rα:gp130        receptor complex;    -   Inhibition of interaction between IL-11:IL-11Rα complex and        gp130.

These properties can be determined by analysis of the relevant agent ina suitable assay, which may involve comparison of the performance of theagent to suitable control agents. The skilled person is able to identifyan appropriate control conditions for a given assay.

For example, a suitable negative control for the analysis of the abilityof a test antibody/antigen-binding fragment to bind to IL-11/an IL-11containing complex/a receptor for IL-11 may be anantibody/antigen-binding fragment directed against a non-target protein(i.e. an antibody/antigen-binding fragment which is not specific forIL-11/an IL-11 containing complex/a receptor for IL-11). A suitablepositive control may be a known, validated (e.g. commercially available)IL-11- or IL-11 receptor-binding antibody. Controls may be of the sameisotype as the putative IL-11/IL-11 containing complex/IL-11receptor-binding antibody/antigen-binding fragment being analysed, andmay e.g. have the same constant regions.

In some embodiments, the agent may be capable of binding specifically toIL-11 or an IL-11 containing complex, or a receptor for IL-11 (e.g.IL-11Rα, gp130, or a complex containing IL-11Rα and/or gp130). An agentwhich specifically binds to a given target molecule preferably binds thetarget with greater affinity, and/or with greater duration than it bindsto other, non-target molecules.

In some embodiments the agent may bind to IL-11 or an IL-11 containingcomplex with greater affinity than the affinity of binding to one ormore other members of the IL-6 cytokine family (e.g. IL-6, leukemiainhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1 (CT-1),ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine(CLC)). In some embodiments the agent may bind to a receptor for IL-11(e.g. IL-11R, gp130, or a complex containing IL-11Rα and/or gp130) withgreater affinity than the affinity of binding to one or more othermembers of the IL-6 receptor family. In some embodiments the agent maybind with greater affinity to IL-11Rα than the affinity of binding toone or more of IL-6Rα, leukemia inhibitory factor receptor (LIFR),oncostatin M receptor (OSMR), ciliary neurotrophic factor receptor alpha(CNTFRα) and cytokine receptor-like factor 1 (CRLF1).

In some embodiments, the extent of binding of a binding agent to annon-target is less than about 10% of the binding of the agent to thetarget as measured, e.g., by ELISA, SPR, Bio-Layer Interferometry (BLI),MicroScale Thermophoresis (MST), or by a radioimmunoassay (RIA).Alternatively, the binding specificity may be reflected in terms ofbinding affinity, where the binding agent binds to IL-11, an IL-11containing complex or a receptor for IL-11 with a K_(D) that is at least0.1 order of magnitude (i.e. 0.1×10n, where n is an integer representingthe order of magnitude) greater than the K_(D) towards another,non-target molecule. This may optionally be one of at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

Binding affinity for a given binding agent for its target is oftendescribed in terms of its dissociation constant (K_(D)). Bindingaffinity can be measured by methods known in the art, such as by ELISA,Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol(2012) 907:411-442; or Rich et al., Anal Biochem. 2008 Feb. 1;373(1):112-20), Bio-Layer Interferometry (see e.g. Lad et al., (2015) JBiomol Screen 20(4): 498-507; or Concepcion et al., Comb Chem HighThroughput Screen. 2009 September; 12(8):791-800), MicroScaleThermophoresis (MST) analysis (see e.g. Jerabek-Willemsen et al., AssayDrug Dev Technol. 2011 August; 9(4): 342-353), or by a radiolabelledantigen binding assay (RIA).

In some embodiments, the agent is capable of binding to IL-11 or anIL-11 containing complex, or a receptor for IL-11 with a K_(D) of 50 μMor less, preferably one of ≤10 μM, ≤5 μM, ≤4 μM, ≤3 μM, ≤2 μM, ≤1 μM,≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM 53 nM, ≤2 nM, ≤1nM, ≤500 μM, ≤400 μM, ≤300 μM, ≤200 μM, or 5100 μM.

In some embodiments, the agent binds to IL-11, an IL-11 containingcomplex or a receptor for IL-11 with an affinity of binding (e.g. asdetermined by ELISA) of EC50=10,000 ng/ml or less, preferably one of≤5,000 ng/ml, ≤1000 ng/ml, ≤900 ng/ml, ≤800 ng/ml, ≤700 ng/ml, ≤600ng/ml, ≤500 ng/ml, ≤400 ng/ml, ≤300 ng/ml, 5200 ng/ml, ≤100 ng/ml, ≤90ng/ml, ≤80 ng/ml, ≤70 ng/ml, ≤60 ng/ml, ≤50 ng/ml, 40 ng/ml, 530 ng/ml,≤20 ng/ml, ≤15 ng/ml, ≤10 ng/ml, ≤7.5 ng/ml, ≤5 ng/ml, ≤2.5 ng/ml, or ≤1ng/ml. Such ELISAs can be performed e.g. as described in AntibodyEngineering, vol. 1 (2nd Edn). Springer Protocols. Springer (2010), PartV, pp 657-665.

In some embodiments, the agent binds to IL-11 or an IL-11-containingcomplex in a region which is important for binding to a receptor for theIL-11 or IL-11-containing complex, e.g. gp130 or IL-11Rα, and therebyinhibits interaction between IL-11 or an IL-11-containing complex and areceptor for IL-11, and/or signalling through the receptor. In someembodiments, the agent binds to a receptor for IL-11 in a region whichis important for binding to IL-11 or an IL-11-containing complex, andthereby inhibits interaction between IL-11 or an IL-11-containingcomplex and a receptor for IL-11, and/or signalling through thereceptor.

The ability of a given binding agent (e.g. an agent capable of bindingIL-11/an IL-11 containing complex or a receptor for IL-11) to inhibitinteraction between two proteins can be determined for example byanalysis of interaction in the presence of, or following incubation ofone or both of the interaction partners with, the binding agent. Anexample of a suitable assay to determine whether a given binding agentis capable of inhibiting interaction between two interaction partners isa competition ELISA.

A binding agent which is capable of inhibiting a given interaction (e.g.between IL-11 and IL-11Rα, or between IL-11 and gp130, or between IL-11and IL-11Rα:gp130, or between IL-11:IL-11Rα and gp130) is identified bythe observation of a reduction/decrease in the level of interactionbetween the interaction partners in the presence of—or followingincubation of one or both of the interaction partners with—the bindingagent, as compared to the level of interaction in the absence of thebinding agent (or in the presence of an appropriate control bindingagent). Suitable analysis can be performed in vitro, e.g. usingrecombinant interaction partners or using cells expressing theinteraction partners. Cells expressing interaction partners may do soendogenously, or may do so from nucleic acid introduced into the cell.For the purposes of such assays, one or both of the interaction partnersand/or the binding agent may be labelled or used in conjunction with adetectable entity for the purposes of detecting and/or measuring thelevel of interaction. For example, the agent may be labelled with aradioactive atom or a coloured molecule or a fluorescent molecule or amolecule which can be readily detected in any other way. Suitabledetectable molecules include fluorescent proteins, luciferase, enzymesubstrates, and radiolabels. The binding agent may be directly labelledwith a detectable label or it may be indirectly labelled. For example,the binding agent may be unlabelled, and detected by another bindingagent which is itself labelled.

Alternatively, the second binding agent may have bound to it biotin andbinding of labelled streptavidin to the biotin may be used to indirectlylabel the first binding agent.

Ability of a binding agent to inhibit interaction between two bindingpartners can also be determined by analysis of the downstream functionalconsequences of such interaction, e.g. IL-11-mediated signalling. Forexample, downstream functional consequences of interaction between IL-11and IL-11Rα:gp130 or between IL-11:IL-11Rα and gp130 may include e.g. aprocess mediated by IL-11, or gene/protein expression of e.g. collagenor IL-11.

Inhibition of interaction between IL-11 or an IL-11 containing complexand a receptor for IL-11 can be analysed using 3H-thymidineincorporation and/or Ba/F3 cell proliferation assays such as thosedescribed in e.g. Curtis et al. Blood, 1997, 90(11) and Karpovich et al.Mol. Hum. Reprod. 2003 9(2): 75-80. Ba/F3 cells co-express IL-11Rα andgp130.

In some embodiments, the binding agent may be capable of inhibitinginteraction between IL-11 and IL-11Rα to less than 100%, e.g. one of 99%or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% orless, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less,40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% orless, 10% or less, 5% or less, or 1% or less of the level of interactionbetween IL-11 and IL-11Rα in the absence of the binding agent (or in thepresence of an appropriate control binding agent). In some embodiments,the binding agent may be capable of inhibiting interaction between IL-11and IL-11Rα to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times,≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times,≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1times the level of interaction between IL-11 and IL-11Rα in the absenceof the binding agent (or in the presence of an appropriate controlbinding agent).

In some embodiments, the binding agent may be capable of inhibitinginteraction between IL-11 and gp130 to less than 100%, e.g. one of 99%or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% orless, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less,40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% orless, 10% or less, 5% or less, or 1% or less of the level of interactionbetween IL-11 and gp130 in the absence of the binding agent (or in thepresence of an appropriate control binding agent). In some embodiments,the binding agent may be capable of inhibiting interaction between IL-11and gp130 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times,≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times,≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1times the level of interaction between IL-11 and gp130 in the absence ofthe binding agent (or in the presence of an appropriate control bindingagent).

In some embodiments, the binding agent may be capable of inhibitinginteraction between IL-11 and IL-11Rα:gp130 to less than 100%, e.g. oneof 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70%or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% orless, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less,15% or less, 10% or less, 5% or less, or 1% or less of the level ofinteraction between IL-11 and IL-11Rα:gp130 in the absence of thebinding agent (or in the presence of an appropriate control bindingagent). In some embodiments, the binding agent may be capable ofinhibiting interaction between IL-11 and IL-11Rα:gp130 to less than 1times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times,≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times,≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times the level ofinteraction between IL-11 and IL-11Rα:gp130 in the absence of thebinding agent (or in the presence of an appropriate control bindingagent).

In some embodiments, the binding agent may be capable of inhibitinginteraction between IL-11:IL-11Rα complex and gp130 to less than 100%,e.g. one of 99% or less, 95% or less, 90% or less, 85% or less, 75% orless, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less,45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% orless, 15% or less, 10% or less, 5% or less, or 1% or less of the levelof interaction between IL-11:IL-11Rα complex and gp130 in the absence ofthe binding agent (or in the presence of an appropriate control bindingagent). In some embodiments, the binding agent is capable of inhibitinginteraction between IL-11:IL-11Rα complex and gp130 to less than 1times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times,≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times,≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times the level ofinteraction between IL-11:IL-11Rα complex and gp130 in the absence ofthe binding agent.

Agents Capable of Reducing Expression of IL-11 or an IL-11 Receptor

In aspects of the present invention the agent capable of inhibitingIL-11-mediated signalling may be capable of preventing or reducing theexpression of one or more of IL-11, IL-11Rα or gp130.

Expression may be gene or protein expression, and may be determined asdescribed herein or by methods in the art that will be well known to askilled person. Expression may be by a cell/tissue/organ/organ system ofa subject.

Suitable agents may be of any kind, but in some embodiments an agentcapable of preventing or reducing the expression of one or more ofIL-11, IL-11Rα or gp130 may be a small molecule or an oligonucleotide.

An agent capable of preventing or reducing of the expression of one ormore of IL-11, IL-11Rα or gp130 may do so e.g. through inhibitingtranscription of the gene encoding IL-11, IL-11Rα or gp130, inhibitingpost-transcriptional processing of RNA encoding IL-11, IL-11Rα or gp130,reducing the stability of RNA encoding IL-11, IL-11Rα or gp130,promoting degradation of RNA encoding IL-11, IL-11Rα or gp130,inhibiting post-translational processing of IL-11, IL-11Rα or gp130polypeptide, reducing the stability of IL-11, IL-11Rα or gp130polypeptide or promoting degradation of IL-11, IL-11Rα or gp130polypeptide.

Taki et al. Clin Exp Immunol (1998) April; 112(1): 133-138 reported areduction in the expression of IL-11 in rheumatoid synovial cells upontreatment with indomethacin, dexamethasone or interferon-gamma (IFNγ).

The present invention contemplates the use of antisense nucleic acid toprevent/reduce expression of IL-11, IL-11Rα or gp130. In someembodiments, an agent capable of preventing or reducing the expressionof IL-11, IL-11Rα or gp130 may cause reduced expression by RNAinterference (RNAi).

In some embodiments, the agent may be an inhibitory nucleic acid, suchas antisense or small interfering RNA, including but not limited toshRNA or siRNA.

In some embodiments the inhibitory nucleic acid is provided in a vector.For example, in some embodiments the agent may be a lentiviral vectorencoding shRNA for one or more of IL-11, IL-11Rα or gp130.

Oligonucleotide molecules, particularly RNA, may be employed to regulategene expression. These include antisense oligonucleotides, targeteddegradation of mRNAs by small interfering RNAs (siRNAs), posttranscriptional gene silencing (PTGs), developmentally regulatedsequence-specific translational repression of mRNA by micro-RNAs(miRNAs) and targeted transcriptional gene silencing.

An antisense oligonucleotide is an oligonucleotide, preferablysingle-stranded, that targets and binds, by complementary sequencebinding, to a target oligonucleotide, e.g. mRNA. Where the targetoligonucleotide is an mRNA, binding of the antisense to the mRNA blockstranslation of the mRNA and expression of the gene product. Antisenseoligonucleotides may be designed to bind sense genomic nucleic acid andinhibit transcription of a target nucleotide sequence.

In view of the known nucleic acid sequences for IL-11, IL-11Rα and gp130(e.g. the known mRNA 25 sequences available from GenBank under AccessionNo.s: BC012506.1 G:15341754 (human IL-11), BC134354.1 G:126632002 (mouseIL-11), AF347935.1 GI:13549072 (rat IL-11), NM_001142784.2 GI:391353394(human IL-11Rα), NM_001163401.1 GI:254281268 (mouse IL-11Rα),NM_139116.1 GI:20806172 (rat IL-11Rα), NM_001190981.1 GI:300244534(human gp130), NM_010560.3 GI:225007624 (mouse gp130), NM_001008725.3GI:300244570 (rat gp130)) oligonucleotides may be designed to repress orsilence the expression of IL-11, IL-11Rα or gp130.

Such oligonucleotides may have any length, but may preferably be short,e.g. less than 100 nucleotides, e.g. 10-40 nucleotides, or 20-50nucleotides, and may comprise a nucleotide sequence having complete- ornear-complementarity (e.g. 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% complementarity) to a sequence of nucleotides ofcorresponding length in the target oligonucleotide, e.g. the IL-11,IL-11Rα or gp130 mRNA. The complementary region of the nucleotidesequence may have any length, but is preferably at least 5, andoptionally no more than 50, nucleotides long, e.g. one of 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, or 50 nucleotides.

Repression of expression of IL-11, IL-11Rα or gp130 will preferablyresult in a decrease in the quantity of IL-11, IL-11Rα or gp130expressed by a cell/tissue/organ/organ system/subject. For example, in agiven cell the repression of IL-11, IL-11Rα or gp130 by administrationof a suitable nucleic acid will result in a decrease in the quantity ofIL-11, IL-11Rα or gp130 expressed by that cell relative to an untreatedcell. Repression may be partial. Preferred degrees of repression are atleast 50%, more preferably one of at least 60%, 70%, 80%, 85% or 90%. Alevel of repression between 90% and 100% is considered a ‘silencing’ ofexpression or function.

A role for the RNAi machinery and small RNAs in targeting ofheterochromatin complexes and epigenetic gene silencing at specificchromosomal loci has been demonstrated. Double-stranded RNA(dsRNA)-dependent post transcriptional silencing, also known as RNAinterference (RNAi), is a phenomenon in which dsRNA complexes can targetspecific genes of homology for silencing in a short period of time. Itacts as a signal to promote degradation of mRNA with sequence identity.A 20-nt siRNA is generally long enough to induce gene-specificsilencing, but short enough to evade host response. The decrease inexpression of targeted gene products can be extensive with 90% silencinginduced by a few molecules of siRNA. RNAi based therapeutics have beenprogressed into Phase I, II and III clinical trials for a number ofindications (Nature 2009 Jan. 22; 457(7228):426-433).

In the art, these RNA sequences are termed “short or small interferingRNAs” (siRNAs) or “microRNAs” (miRNAs) depending on their origin. Bothtypes of sequence may be used to down-regulate gene expression bybinding to complementary RNAs and either triggering mRNA elimination(RNAi) or arresting mRNA translation into protein. siRNA are derived byprocessing of long double stranded RNAs and when found in nature aretypically of exogenous origin. Micro-interfering RNAs (miRNA) areendogenously encoded small non-coding RNAs, derived by processing ofshort hairpins. Both siRNA and miRNA can inhibit the translation ofmRNAs bearing partially complimentary target sequences without RNAcleavage and degrade mRNAs bearing fully complementary sequences.

siRNA ligands are typically double stranded and, in order to optimisethe effectiveness of RNA mediated down-regulation of the function of atarget gene, it is preferred that the length of the siRNA molecule ischosen to ensure correct recognition of the siRNA by the RISC complexthat mediates the recognition by the siRNA of the mRNA target and sothat the siRNA is short enough to reduce a host response.

miRNA ligands are typically single stranded and have regions that arepartially complementary enabling the ligands to form a hairpin. miRNAsare RNA genes which are transcribed from DNA, but are not translatedinto protein. A DNA sequence that codes for a miRNA gene is longer thanthe miRNA. This DNA sequence includes the miRNA sequence and anapproximate reverse complement. When this DNA sequence is transcribedinto a single-stranded RNA molecule, the miRNA sequence and itsreverse-complement base pair to form a partially double stranded RNAsegment. The design of microRNA sequences is discussed in John et al,PLoS Biology, 11(2), 1862-1879, 2004.

Typically, the RNA ligands intended to mimic the effects of siRNA ormiRNA have between 10 and 40 ribonucleotides (or synthetic analoguesthereof), more preferably between 17 and 30 ribonucleotides, morepreferably between 19 and 25 ribonucleotides and most preferably between21 and 23 ribonucleotides. In some embodiments of the inventionemploying double-stranded siRNA, the molecule may have symmetric 3′overhangs, e.g. of one or two (ribo)nucleotides, typically a UU of dTdT3′ overhang. Based on the disclosure provided herein, the skilled personcan readily design suitable siRNA and miRNA sequences, for example usingresources such the Ambion siRNA finder. siRNA and miRNA sequences can besynthetically produced and added exogenously to cause genedownregulation or produced using expression systems (e.g. vectors). In apreferred embodiment the siRNA is synthesized synthetically.

Longer double stranded RNAs may be processed in the cell to producesiRNAs (see for example Myers (2003) Nature Biotechnology 21:324-328).The longer dsRNA molecule may have symmetric 3′ or 5′ overhangs, e.g. ofone or two (ribo)nucleotides, or may have blunt ends. The longer dsRNAmolecules may be 25 nucleotides or longer. Preferably, the longer dsRNAmolecules are between 25 and 30 nucleotides long. More preferably, thelonger dsRNA molecules are between 25 and 27 nucleotides long. Mostpreferably, the longer dsRNA molecules are 27 nucleotides in length.dsRNAs 30 nucleotides or more in length may be expressed using thevector pDECAP (Shinagawa et al., Genes and Dev., 17, 1340-5, 2003).

Another alternative is the expression of a short hairpin RNA molecule(shRNA) in the cell. shRNAs are more stable than synthetic siRNAs. AshRNA consists of short inverted repeats separated by a small loopsequence. One inverted repeat is complimentary to the gene target. Inthe cell the shRNA is processed by DICER into a siRNA which degrades thetarget gene mRNA and suppresses expression. In a preferred embodimentthe shRNA is produced endogenously (within a cell) by transcription froma vector. shRNAs may be produced within a cell by transfecting the cellwith a vector encoding the shRNA sequence under control of a RNApolymerase III promoter such as the human H1 or 7SK promoter or a RNApolymerase II promoter. Alternatively, the shRNA may be synthesisedexogenously (in vitro) by transcription from a vector. The shRNA maythen be introduced directly into the cell. Preferably, the shRNAmolecule comprises a partial sequence of IL-11, IL-11Rα or gp130.Preferably, the shRNA sequence is between 40 and 100 bases in length,more preferably between 40 and 70 bases in length. The stem of thehairpin is preferably between 19 and 30 base pairs in length. The stemmay contain G-U pairings to stabilise the hairpin structure.

siRNA molecules, longer dsRNA molecules or miRNA molecules may be maderecombinantly by transcription of a nucleic acid sequence, preferablycontained within a vector. Preferably, the siRNA molecule, longer dsRNAmolecule or miRNA molecule comprises a partial sequence of IL-11,IL-11Rα or gp130.

In one embodiment, the siRNA, longer dsRNA or miRNA is producedendogenously (within a cell) by transcription from a vector. The vectormay be introduced into the cell in any of the ways known in the art.Optionally, expression of the RNA sequence can be regulated using atissue specific (e.g. heart, liver, or kidney specific) promoter. In afurther embodiment, the siRNA, longer dsRNA or miRNA is producedexogenously (in vitro) by transcription from a vector.

Suitable vectors may be oligonucleotide vectors configured to expressthe oligonucleotide agent capable of IL-11, IL-11Rα or gp130 repression.Such vectors may be viral vectors or plasmid vectors. The therapeuticoligonucleotide may be incorporated in the genome of a viral vector andbe operably linked to a regulatory sequence, e.g. promoter, which drivesits expression. The term “operably linked” may include the situationwhere a selected nucleotide sequence and regulatory nucleotide sequenceare covalently linked in such a way as to place the expression of anucleotide sequence under the influence or control of the regulatorysequence. Thus a regulatory sequence is operably linked to a selectednucleotide sequence if the regulatory sequence is capable of effectingtranscription of a nucleotide sequence which forms part or all of theselected nucleotide sequence.

Viral vectors encoding promoter-expressed siRNA sequences are known inthe art and have the benefit of long term expression of the therapeuticoligonucleotide. Examples include lentiviral (Nature 2009 Jan. 22;457(7228):426-433), adenovirus (Shen et al., FEBS Lett 2003 Mar. 27;539(1-3)111-4) and retroviruses (Barton and Medzhitov PNAS Nov. 12, 2002vol. 99, no. 23 14943-14945).

In other embodiments a vector may be configured to assist delivery ofthe therapeutic oligonucleotide to the site at which repression ofIL-11. IL-11Rα or gp130 expression is required. Such vectors typicallyinvolve complexing the oligonucleotide with a positively charged vector(e.g., cationic cell penetrating peptides, cationic polymers anddendrimers, and cationic lipids): conjugating the oligonucleotide withsmall molecules (e.g., cholesterol, bile acids, and lipids), polymers,antibodies, and RNAs; or encapsulating the oligonucleotide innanoparticulate formulations (Wang et al., AAPS J. 2010 December; 12(4):492-503).

In one embodiment, a vector may comprise a nucleic acid sequence in boththe sense and antisense orientation, such that when expressed as RNA thesense and antisense sections will associate to form a double strandedRNA.

Alternatively, siRNA molecules may be synthesized using standard solidor solution phase synthesis techniques which are known in the art.Linkages between nucleotides may be phosphodiester bonds oralternatives, for example, linking groups of the formula P(O)S,(thioate); P(S)S, (dithioate); P(O)NR′2; P(O)R′; P(O)OR6; CO; or CONR′2wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) isjoined to adjacent nucleotides through-O-or-S—.

Modified nucleotide bases can be used in addition to the naturallyoccurring bases, and may confer advantageous properties on siRNAmolecules containing them.

For example, modified bases may increase the stability of the siRNAmolecule, thereby reducing the amount required for silencing. Theprovision of modified bases may also provide siRNA molecules which aremore, or less, stable than unmodified siRNA.

The term ‘modified nucleotide base’ encompasses nucleotides with acovalently modified base and/or sugar. For example, modified nucleotidesinclude nucleotides having sugars which are covalently attached to lowmolecular weight organic groups other than a hydroxyl group at the3′position and other than a phosphate group at the 5′position. Thusmodified nucleotides may also include 2′substituted sugars such as2′-O-methyl-; 2′-O-alkyl; 2′-O-allyl; 2′-S-alkyl; 2′-S-allyl;2′-fluoro-; 2′-halo or azido-ribose, carbocyclic sugar analogues,a-anomeric sugars; epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, and sedoheptulose.

Modified nucleotides are known in the art and include alkylated purinesand pyrimidines, acylated purines and pyrimidines, and otherheterocycles. These classes of pyrimidines and purines are known in theart and include pseudoisocytosine, N4,N4-ethanocytosine,8-hydroxy-N6-methyladenine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil, 5 fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1-methyladenine,1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine,2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine,N6-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxyamino methyl-2-thiouracil, -D-mannosylqueosine,5-methoxycarbonylmethyluracil, 5methoxyuracil, 2methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester,pseudouracil, 2-thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil,4-thiouracil, 5methyluracil, N-uracil-5-oxyacetic acid methylester,uracil 5-oxyacetic acid, queosine, 2-thiocytosine, 5-propyluracil,5-propylcytosine, 5-ethyluracil, 5ethylcytosine, 5-butyluracil,5-phenyluracil, 5-pentylcytosine, and 2,6,diaminopurine,methylpsuedouracil, 1-methylguanine, 1-methylcytosine.

Methods relating to the use of RNAi to silence genes in C. elegans,Drosophila, plants, and mammals are known in the art (Fire A, et al.,1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999);Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001);Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119 (2001); Tuschl,T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A. et al., Science 286,950-952 (1999); Hammond, S. M., et al., Nature 404, 293-296 (2000);Zamore, P. D., et al., Cell 101, 25-33 (2000): Bernstein, E., et al.,Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15,188-200 (2001); WO0129058; WO9932619, and Elbashir S M, et al., 2001Nature 411:494-498).

Accordingly, the invention provides nucleic acid that is capable, whensuitably introduced into or expressed within a mammalian, e.g. human,cell that otherwise expresses IL-11, IL-11Rα or gp130, of suppressingIL-11, IL-11Rα or gp130 expression by RNAi.

Nucleic acid sequences for IL-11, IL-11Rα and gp130 (e.g. the known mRNAsequences available from GenBank under Accession No.s: BC012506.1GI:15341754 (human IL-11), BC134354.1 GI:126632002 (mouse IL-11),AF347935.1 GI:13549072 (rat IL-11), NM_001142784.2 GI:391353394 (humanIL-11Rα), NM_001163401.1 GI:254281268 (mouse IL-11Rα), NM_139116.1GI:20806172 (rat IL-11Rα), NM_001190981.1 GI:300244534 (human gp130),NM_010560.3 GI:225007624 (mouse gp130), NM_001008725.3 GI:300244570 (ratgp130)) oligonucleotides may be designed to repress or silence theexpression of IL-11, IL-11Rα or gp130.

The nucleic acid may have substantial sequence identity to a portion ofIL-11, IL-11Rα or gp130 mRNA, e.g. as defined in GenBank accession no.NM_000641.3 GI:391353405 (IL-11), NM_001142784.2 GI:391353394 (IL-11Rα),NM_001190981.1 GI:300244534 (gp130) or the complementary sequence tosaid mRNA.

The nucleic acid may be a double-stranded siRNA. (As the skilled personwill appreciate, and as explained further below, a siRNA molecule mayinclude a short 3′ DNA sequence also.)

Alternatively, the nucleic acid may be a DNA (usually double-strandedDNA) which, when transcribed in a mammalian cell, yields an RNA havingtwo complementary portions joined via a spacer, such that the RNA takesthe form of a hairpin when the complementary portions hybridise witheach other. In a mammalian cell, the hairpin structure may be cleavedfrom the molecule by the enzyme DICER, to yield two distinct, buthybridised. RNA molecules.

In some preferred embodiments, the nucleic acid is generally targeted tothe sequence of one of SEQ ID NOs 4 to 7 (IL-11) or to one of SEQ ID NOs8 to 11 (IL-11Rα).

Only single-stranded (i.e. non self-hybridised) regions of an mRNAtranscript are expected to be suitable targets for RNAi. It is thereforeproposed that other sequences very close in the IL-11 or IL-11Rα mRNAtranscript to the sequence represented by one of SEQ ID NOs 4 to 7 or 8to 11 may also be suitable targets for RNAi. Such target sequences arepreferably 17-23 nucleotides in length and preferably overlap one of SEQID NOs 4 to 7 or 8 to 11 by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18 or all 19 nucleotides (at either end of oneof SEQ ID NOs 4 to 7 or 8 to 11).

Accordingly, the invention provides nucleic acid that is capable, whensuitably introduced into or expressed within a mammalian cell thatotherwise expresses IL-11 or IL-11Rα, of suppressing IL-11 or IL-11Rαexpression by RNAi, wherein the nucleic acid is generally targeted tothe sequence of one of SEQ ID NOs 4 to 7 or 8 to 11.

By “generally targeted” the nucleic acid may target a sequence thatoverlaps with SEQ ID NOs 4 to 7 or 8 to 11. In particular, the nucleicacid may target a sequence in the mRNA of human IL-11 or IL-11Rα that isslightly longer or shorter than one of SEQ ID NOs 4 to 7 or 8 to 11(preferably from 17-23 nucleotides in length), but is otherwiseidentical to one of SEQ ID NOs 4 to 7 or 8 to 11.

It is expected that perfect identity/complementarity between the nucleicacid of the invention and the target sequence, although preferred, isnot essential. Accordingly, the nucleic acid of the invention mayinclude a single mismatch compared to the mRNA of IL-11 or IL-11Rα. Itis expected, however, that the presence of even a single mismatch islikely to lead to reduced efficiency, so the absence of mismatches ispreferred. When present, 3′ overhangs may be excluded from theconsideration of the number of mismatches.

The term “complementarity” is not limited to conventional base pairingbetween nucleic acid consisting of naturally occurring ribo- and/ordeoxyribonucleotides, but also includes base pairing between mRNA andnucleic acids of the invention that include non-natural nucleotides.

In one embodiment, the nucleic acid (herein referred to asdouble-stranded siRNA) includes the double-stranded RNA sequences shownin SEQ ID NOs 12 to 15. In another embodiment, the nucleic acid (hereinreferred to as double-stranded siRNA) includes the double-stranded RNAsequences shown in SEQ ID NOs 16 to 19.

However, it is also expected that slightly shorter or longer sequencesdirected to the same region of IL-11 or IL-11Rα mRNA will also beeffective. In particular, it is expected that double-stranded sequencesbetween 17 and 23 bp in length will also be effective.

The strands that form the double-stranded RNA may have short 3′dinucleotide overhangs, which may be DNA or RNA. The use of a 3′ DNAoverhang has no effect on siRNA activity compared to a 3′ RNA overhang,but reduces the cost of chemical synthesis of the nucleic acid strands(Elbashir et al., 2001c). For this reason, DNA dinucleotides may bepreferred.

When present, the dinucleotide overhangs may be symmetrical to eachother, though this is not essential. Indeed, the 3′ overhang of thesense (upper) strand is irrelevant for RNAi activity, as it does notparticipate in mRNA recognition and degradation (Elbashir et al., 2001a,2001b, 2001c).

While RNAi experiments in Drosophila show that antisense 3′ overhangsmay participate in mRNA recognition and targeting (Elbashir et al.2001c), 3′ overhangs do not appear to be necessary for RNAi activity ofsiRNA in mammalian cells. Incorrect annealing of 3′ overhangs istherefore thought to have little effect in mammalian cells (Elbashir etal. 2001c; Czauderna et al. 2003).

Any dinucleotide overhang may therefore be used in the antisense strandof the siRNA. Nevertheless, the dinucleotide is preferably -UU or -UG(or -TT or -TG if the overhang is DNA), more preferably -UU (or -TT).The -UU (or -TT) dinucleotide overhang is most effective and isconsistent with (i.e. capable of forming part of) the RNA polymerase IIIend of transcription signal (the terminator signal is TTTTT).Accordingly, this dinucleotide is most preferred. The dinucleotides AA,CC and GG may also be used, but are less effective and consequently lesspreferred.

Moreover, the 3′ overhangs may be omitted entirely from the siRNA.

The invention also provides single-stranded nucleic acids (hereinreferred to as single-stranded siRNAs) respectively consisting of acomponent strand of one of the aforementioned double-stranded nucleicacids, preferably with the 3′-overhangs, but optionally without. Theinvention also provides kits containing pairs of such single-strandednucleic acids, which are capable of hybridising with each other in vitroto form the aforementioned double-stranded siRNAs, which may then beintroduced into cells.

The invention also provides DNA that, when transcribed in a mammaliancell, yields an RNA (herein also referred to as an shRNA) having twocomplementary portions which are capable of self-hybridising to producea double-stranded motif, e.g. including a sequence selected from thegroup consisting of SEQ ID NOs: 12 to 15 or 16 to 19 or a sequence thatdiffers from any one of the aforementioned sequences by a single basepair substitution.

The complementary portions will generally be joined by a spacer, whichhas suitable length and sequence to allow the two complementary portionsto hybridise with each other. The two complementary (i.e. sense andantisense) portions may be joined 5′-3′ in either order. The spacer willtypically be a short sequence, of approximately 4-12 nucleotides,preferably 4-9 nucleotides, more preferably 6-9 nucleotides.

Preferably the 5′ end of the spacer (immediately 3′ of the upstreamcomplementary portion) consists of the nucleotides -UU- or -UG-, againpreferably -UU- (though, again, the use of these particulardinucleotides is not essential). A suitable spacer, recommended for usein the pSuper system of OligoEngine (Seattle, Wash., USA) is UUCAAGAGA.In this and other cases, the ends of the spacer may hybridise with eachother. e.g. elongating the double-stranded motif beyond the exactsequences of SEQ ID NOs 12 to 15 or 16 to 19 by a small number (e.g. 1or 2) of base pairs.

Similarly, the transcribed RNA preferably includes a3′ overhang from thedownstream complementary portion. Again, this is preferably -UU or -UG,more preferably -UU.

Such shRNA molecules may then be cleaved in the mammalian cell by theenzyme DICER to yield a double-stranded siRNA as described above, inwhich one or each strand of the hybridised dsRNA includes a 3′ overhang.

Techniques for the synthesis of the nucleic acids of the invention areof course well known in the art.

The skilled person is well able to construct suitable transcriptionvectors for the DNA of the invention using well-known techniques andcommercially available materials. In particular, the DNA will beassociated with control sequences, including a promoter and atranscription termination sequence.

Of particular suitability are the commercially available pSuper andpSuperior systems of OligoEngine (Seattle, Wash., USA). These use apolymerase-III promoter (H1) and a T5 transcription terminator sequencethat contributes two U residues at the 3′ end of the transcript (which,after DICER processing, provide a 3′ UU overhang of one strand of thesiRNA).

Another suitable system is described in Shin et al. (RNA, 2009 May;15(5): 898-910), which uses another polymerase-III promoter (U6).

The double-stranded siRNAs of the invention may be introduced intomammalian cells in vitro or in vivo using known techniques, as describedbelow, to suppress expression of IL-11 or a receptor for IL-11.

Similarly, transcription vectors containing the DNAs of the inventionmay be introduced into tumour cells in vitro or in vivo using knowntechniques, as described below, for transient or stable expression ofRNA, again to suppress expression of IL-11 or a receptor for IL-11.

Accordingly, the invention also provides a method of suppressingexpression of IL-11 or a receptor for IL-11 in a mammalian, e.g. human,cell, the method comprising administering to the cell a double-strandedsiRNA of the invention or a transcription vector of the invention.

Similarly, the invention further provides a method of treating kidneyinjury and/or a disorder, disease or condition associated with kidneyinjury, the method comprising administering to a subject adouble-stranded siRNA of the invention or a transcription vector of theinvention.

The invention further provides the double-stranded siRNAs of theinvention and the transcription vectors of the invention, for use in amethod of treatment, preferably a method of treating kidney injuryand/or a disorder, disease or condition associated with kidney injury.

The invention further provides the use of the double-stranded siRNAs ofthe invention and the transcription vectors of the invention in thepreparation of a medicament for the treatment of kidney injury and/or adisorder, disease or condition associated with kidney injury.

The invention further provides a composition comprising adouble-stranded siRNA of the invention or a transcription vector of theinvention in admixture with one or more pharmaceutically acceptablecarriers.

Suitable carriers include lipophilic carriers or vesicles, which mayassist in penetration of the cell membrane.

Materials and methods suitable for the administration of siRNA duplexesand DNA vectors of the invention are well known in the art and improvedmethods are under development, given the potential of RNAi technology.

Generally, many techniques are available for introducing nucleic acidsinto mammalian cells. The choice of technique will depend on whether thenucleic acid is transferred into cultured cells in vitro or in vivo inthe cells of a patient. Techniques suitable for the transfer of nucleicacid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE, dextran and calciumphosphate precipitation. In vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection (Dzau et al. (2003) Trends inBiotechnology 11, 205-210).

In particular, suitable techniques for cellular administration of thenucleic acids of the invention both in vitro and in vivo are disclosedin the following articles:

General reviews: Borkhardt, A. 2002. Blocking oncogenes in malignantcells by RNA interference—new hope for a highly specific cancertreatment? Cancer Cell. 2:167-8. Hannon, G. J. 2002. RNA interference.Nature. 418:244-51. McManus, M. T., and P. A. Sharp. 2002. Genesilencing in mammals by small interfering RNAs. Nat Rev Genet. 3:737-47.Scherr, M., M. A. Morgan, and M. Eder. 2003b. Gene silencing mediated bysmall interfering RNAs in mammalian cells. Curr Med Chem. 10:245-56.Shuey, D. J., D. E. McCallus, and T. Giordano. 2002. RNAi:gene-silencing in therapeutic intervention. Drug Discov Today. 7:1040-6.

Systemic delivery using liposomes: Lewis, D. L., J. E. Hagstrom, A. G.Loomis, J. A. Wolff, and H. Herweijer. 2002. Efficient delivery of siRNAfor inhibition of gene expression in postnatal mice. Nat Genet.32:107-8. Paul, C. P., P. D. Good, I. Winer, and D. R. Engelke. 2002.Effective expression of small interfering RNA in human cells. NatBiotechnol. 20:505-8. Song, E., S. K. Lee, J. Wang, N. Ince, N. Ouyang,J. Min, J. Chen, P. Shankar, and J. Lieberman. 2003. RNA interferencetargeting Fas protects mice from fulminant hepatitis. Nat Med. 9:347-51.Sorensen, D. R., M. Leirdal, and M. Sioud. 2003. Gene silencing bysystemic delivery of synthetic siRNAs in adult mice. J Mol Biol.327:761-6.

Virus mediated transfer: Abbas-Terki, T., W. Blanco-Bose, N. Deglon, W.Pralong, and P. Aebischer. 2002. Lentiviral-mediated RNA interference.Hum Gene Ther. 13:2197-201. Barton, G. M., and R. Medzhitov. 2002.Retroviral delivery of small interfering RNA into primary cells. ProcNatl Acad Sci USA. 99:14943-5. Devroe, E., and P. A. Silver. 2002.Retrovirus-delivered siRNA. BMC Biotechnol. 2:15. Lori, F., P. Guallini,L. Galluzzi, and J. Lisziewicz. 2002. Gene therapy approaches to HIVinfection. Am J Pharmacogenomics. 2:245-52. Matta, H., B. Hozayev, R.Tomar, P. Chugh, and P. M. Chaudhary. 2003. Use of lentiviral vectorsfor delivery of small interfering RNA. Cancer Biol Ther. 2:206-10. Qin,X. F., D. S. An, I. S. Chen, and D. Baltimore. 2003. Inhibiting HIV-1infection in human T cells by lentiviral-mediated delivery of smallinterfering RNA against CCR5. Proc Natl Acad Sci USA. 100:183-8. Scherr,M., K. Battmer, A. Ganser, and M. Eder. 2003a. Modulation of geneexpression by lentiviral-mediated delivery of small interfering RNA.Cell Cycle. 2:251-7. Shen, C., A. K. Buck, X. Liu, M. Winkler, and S. N.Reske. 2003. Gene silencing by adenovirus-delivered siRNA. FEBS Lett.539:111-4.

Peptide delivery: Morris, M. C., L. Chaloin, F. Heitz, and G. Divita.2000. Translocating peptides and proteins and their use for genedelivery. Curr Opin Biotechnol. 11:461-6. Simeoni, F., M. C. Morris, F.Heitz, and G. Divita. 2003. Insight into the mechanism of thepeptide-based gene delivery system MPG: implications for delivery ofsiRNA into mammalian cells. Nucleic Acids Res. 31:2717-24. Othertechnologies that may be suitable for delivery of siRNA to the targetcells are based on nanoparticles or nanocapsules such as those describedin US patent numbers 6,649,192B and 5,843,509B.

Inhibition of IL-11-Mediated Signalling

In embodiments of the present invention, agents capable of inhibitingthe action of IL-11 may possess one or more of the following functionalproperties:

-   -   Inhibition of signalling mediated by IL-11;    -   Inhibition of signalling mediated by binding of IL-11 to        IL-11Rα:gp130 receptor complex;    -   Inhibition of signalling mediated by binding of IL-11:IL-11Rα        complex to gp130 (i.e. IL-11 trans signalling);    -   Inhibition of a process mediated by IL-11;    -   Inhibition of gene/protein expression of IL-11 and/or IL-11Rα.

These properties can be determined by analysis of the relevant agent ina suitable assay, which may involve comparison of the performance of theagent to suitable control agents. The skilled person is able to identifyan appropriate control conditions for a given assay.

IL-11-mediated signalling and/or processes mediated by IL-11 includessignalling mediated by fragments of IL-11 and polypeptide complexescomprising IL-11 or fragments thereof. IL-11-mediated signalling may besignalling mediated by human IL-11 and/or mouse IL-11. Signallingmediated by IL-11 may occur following binding of IL-11 or an IL-11containing complex to a receptor to which IL-11 or said complex binds.

In some embodiments, an agent may be capable of inhibiting thebiological activity of IL-11 or an IL-11-containing complex.

In some embodiments, the agent is an antagonist of one or moresignalling pathways which are activated by signal transduction throughreceptors comprising IL-11Rα and/or gp130, e.g. IL-11Rα:gp130. In someembodiments, the agent is capable of inhibiting signalling through oneor more immune receptor complexes comprising IL-11Rα and/or gp130, e.g.IL-11Rα:gp130. In various aspects of the present invention, an agentprovided herein is capable of inhibiting IL-11-mediated cis and/or transsignalling. In some embodiments in accordance with the various aspectsof the present invention an agent provided herein is capable ofinhibiting IL-11-mediated cis signalling.

In some embodiments, the agent may be capable of inhibitingIL-11-mediated signalling to less than 100%, e.g. one of 99% or less,95% or less, 90% or less, 85% or less, 80/6 or less, 75% or less, 70% orless, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less,40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% orless, 10% or less, 5% or less, or 1% or less of the level of signallingin the absence of the agent (or in the presence of an appropriatecontrol agent). In some embodiments, the agent is capable of reducingIL-11-mediated signalling to less than 1 times, e.g. one of ≤0.99 times,≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times,≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15times, ≤0.1 times the level of signalling in the absence of the agent(or in the presence of an appropriate control agent).

In some embodiments, the IL-11-mediated signalling may be signallingmediated by binding of IL-11 to IL-11Rα:gp130 receptor. Such signallingcan be analysed e.g. by treating cells expressing IL-11Rα and gp130 withIL-11, or by stimulating IL-11 production in cells which express IL-11Rαand gp130.

The IC₅₀ for an agent for inhibition of IL-11-mediated signalling may bedetermined, e.g. by culturing Ba/F3 cells expressing IL-11Rα and gp130in the presence of human IL-11 and the agent, and measuring 3H-thymidineincorporation into DNA. In some embodiments, the agent may exhibit anIC₅₀ of 10 μg/ml or less, preferably one of ≤5 μg/ml, ≤4 μg/ml, ≤3.5μg/ml, ≤3 μg/ml, ≤2 μg/ml, ≤1 μg/ml, ≤0.9 μg/ml, ≤0.8 μg/ml, ≤0.7 μg/ml,≤0.6 μg/ml, or ≤0.5 μg/ml in such an assay.

In some embodiments, the IL-11-mediated signalling may be signallingmediated by binding of IL-11:IL-11Rα complex to gp130. In someembodiments, the IL-11:IL-11Rα complex may be soluble, e.g. complex ofextracellular domain of IL-11Rα and IL-11, or complex of soluble IL-11Rαisoform/fragment and IL-11. In some embodiments, the soluble IL-11Rα isa soluble (secreted) isoform of IL-11Rα, or is the liberated product ofproteolytic cleavage of the extracellular domain of cell membrane boundIL-11Rα.

In some embodiments, the IL-11:IL-11Rα complex may be cell-bound, e.g.complex of cell-membrane bound IL-11Rα and IL-11. Signalling mediated bybinding of IL-11:IL-11Rα complex to gp130 can be analysed by treatingcells expressing gp130 with IL-11:IL-11Rα complex, e.g. recombinantfusion protein comprising IL-11 joined by a peptide linker to theextracellular domain of IL-11Rα, e.g. hyper IL-11. Hyper IL-11 wasconstructed using fragments of IL-11Rα (amino acid residues 1 to 317consisting of domain 1 to 3; UniProtKB: 014626) and IL-11 (amino acidresidues 22 to 199 of UniProtKB: P20809) with a 20 amino acid longlinker (SEQ ID NO:20). The amino acid sequence for Hyper IL-11 is shownin SEQ ID NO:21.

In some embodiments, the agent may be capable of inhibiting signallingmediated by binding of IL-11:IL-11Rα complex to gp130, and is alsocapable of inhibiting signalling mediated by binding of IL-11 toIL-11Rα:gp130 receptor.

In some embodiments, the agent may be capable of inhibiting a processmediated by IL-11.

In some embodiments, the agent may be capable of inhibiting gene/proteinexpression of IL-11 and/or IL-11Rα. Gene and/or protein expression canbe measured as described herein or by methods in the art that will bewell known to a skilled person.

In some embodiments, the agent may be capable of inhibiting gene/proteinexpression of IL-11 and/or IL-11Rα to less than 100%, e.g. one of 99% orless, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less,70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% orless, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less,15% or less, 10% or less, 5% or less, or 1% or less of the level ofexpression in the absence of the agent (or in the presence of anappropriate control agent). In some embodiments, the agent is capable ofinhibiting gene/protein expression of IL-11 and/or IL-11Rα to less than1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times,≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 Limes, 50.55times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times,≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times the level of expressionin the absence of the agent (or in the presence of an appropriatecontrol agent).

Treatment/Prevention of Kidney Injury

The present invention provides methods and articles (agents andcompositions) for the treatment/prevention of kidney injury, e.g. asdescribed herein and disorders, diseases or conditions associated withkidney injury.

Treatment is achieved by inhibition of IL-11-mediating signalling (i.e.antagonism of IL-11-mediated signalling). That is, the present inventionprovides for the treatment/prevention of kidney injury and disorders,diseases and conditions associated with kidney injury through inhibitionof IL-11 mediated signalling, in e.g. a cell, tissue/organ/organsystem/subject. In some embodiments, inhibition of IL-11-mediatedsignalling in accordance with the present disclosure comprisesinhibition of IL-11-mediated signalling in cells of the kidney (e.g.tubule epithelial cells).

Provided is an agent capable of inhibiting interleukin 11(IL-11)-mediated signalling for use in a method of treating orpreventing kidney injury and/or a disorder, disease or conditionassociated with kidney injury.

Also provided is use of an agent capable of inhibiting interleukin 11(IL-11)-mediated signalling for use in the manufacture of a medicamentfor use in a method of treating or preventing kidney injury and/or adisorder, disease or condition associated with kidney injury.

Further provided is a method of treating or preventing kidney injuryand/or a disorder, disease or condition associated with kidney injury,the method comprising administering to a subject in need of treatment atherapeutically effective amount of an agent capable of inhibitinginterleukin 11 (IL-11)-mediated signalling.

In some embodiments, the present invention provides for thetreatment/prevention of kidney injury-related pathology in adisease/condition. That is, the present invention provides for thetreatment/prevention of a disease/condition in which kidney injury ispathologically implicated. Kidney injury-related pathology is describedherein. In particular, relevant pathology includes damage to tubularepithelial cells, either proximal, distal or both.

Agents capable of inhibiting interleukin 11 (IL-11)-mediated signallingare moreover demonstrated herein to be capable of reversing kidneyinjury. That is, inhibition of IL-11 mediated signalling is shown to beable to improve renal function following kidney injury.

Accordingly, the present invention contemplates to employ antagonists ofIL-11 mediated signalling to enhance/improve renal function in subjectshaving impaired renal function. e.g. as a consequence of kidney injury.

Agents capable of inhibiting interleukin 11 (IL-11)-mediated signallingare useful for promoting the proliferation TECs, for the generation offunctional, renal tissue. Thus agents capable of inhibiting interleukin11 (IL-11)-mediated signalling are provided herein for promoting theproliferation, survival and/or function of tubular epithelial cells(TECs), and/or the growth, maintenance and/or function of renal tissue.

Agents capable of inhibiting interleukin 11 (IL-11)-mediated signallingare provided herein for regenerating tubular epithelial cells (TECs),and/or renal tissue.

The epithelial and/or acta to mesenchymal cell phenotype transition(also referred to herein as ‘EMT’) by tubular epithelial cells isassociated with reduced kidney function. EMT by TECs is induced bysoluble factors produced following tissue injury, such as TGFB1.Antagonism of IL-11 mediated signalling is shown herein to inhibit theEMT by TECs, and thereby preserve/improve renal function.

Expression of SNAIL is implicated in the loss of the ability of TECs toproliferate, and EMT. IL-11-mediated signalling is demonstrated hereinto have a central role in upregulating SNAIL expression. Accordingly,the present invention contemplates to employ antagonists of IL-11mediated signalling to inhibit SNAIL expression, e.g. in TECs.Antagonism of IL-11-mediated signalling releases TECs fromSNAIL-mediated inhibition of TEC proliferation.

Accordingly, the present invention contemplates to employ antagonists ofIL-11 mediated signalling to preserve/increase the number/proportion ofcells in the kidney having a TEC phenotype. TEC phenotype may becharacterised e.g. by E-cadherin expression. The present inventioncontemplates to employ antagonists of IL-11 mediated signalling toreduce the number/proportion of cells in the kidney having a mesenchymalcell-like phenotype. A mesenchymal cell-like phenotype may becharacterised e.g. by SNAIL and/or ACTA2 expression, optionally withlack of E-cadherin expression.

Antagonists of IL-11 mediated signalling may be used in methods topreserve/increase the level of a function of TECs or renal tissue.TEC/renal tissue function may be evaluated e.g. by evaluation of acorrelate of such activity. For example, TEC/renal tissue function maybe evaluated by analysing the level of urea and/or creatine in theserum/blood (e.g. blood urea nitrogen), or by monitoring the albumin tocreatine ratio (ACR). Antagonists of IL-11 mediated signalling may beused in methods to reduce the level of creatine and/or urea in theserum/blood, or to reduce ACR.

It will be clear to the person skilled in the art that the therapeuticand prophylactic utility of the present invention extends to essentiallyany disease/condition which would benefit from a reduction in kidneyinjury and/or kidney injury-related pathology. The therapeutic andprophylactic utility of the present invention extends to any subjectsuffering from kidney injury and/or a disorder, disease or conditionassociated with kidney injury. The therapeutic and prophylactic utilityof the present invention also extends to any subject suffering from adisease in which kidney injury-related pathology is present.

In some embodiments, the present invention provides for thetreatment/prevention of diseases/conditions that are caused/exacerbatedby kidney injury. In some embodiments, there is provided thetreatment/prevention of diseases/conditions in a subject in which kidneyinjury provides a poor prognosis.

Diagnosis and management of acute kidney injury is described in Rahman Met al., Acute Kidney injury: a guide to diagnosis and management. Am FamPhysician 2012 Oct. 1:86(7): 631-9. As described therein, acute kidneyinjury is characterized by abrupt deterioration in kidney function,manifested by an increase in serum creatinine level with or withoutreduced urine output.

In some embodiments, kidney injury and/or a disorder, disease orcondition associated with kidney injury to be treated/prevented may becharacterised by an increase in one or more of the following in anorgan/tissue/subject affected by kidney injury and/or a disorder,disease or condition associated with kidney injury e.g. as compared tonormal organ/tissue/subject (i.e. without kidney injury or a disorder,disease or condition associated with kidney injury): expression of oneor more of IL-11, and IL-11Rα.

In some embodiments, the present invention provides for thetreatment/prevention of kidney injury in the context of adisease/disorder/condition associated with kidney injury e.g. asdescribed herein. In some embodiments, the present invention providesfor the treatment/prevention of kidney injury and an underlyingdisease/disorder/condition associated with kidney injury. For example,inhibition of IL-11-mediated signalling has utility in antagonising therole of IL-11 in chemotherapy-associated kidney injury, as well asantagonising the role of IL-11 in the cancer itself.

Treatment/prevention of kidney injury and/or a disorder, disease orcondition associated with kidney injury according to the presentinvention may be of kidney injury and/or a disorder, disease orcondition associated with kidney injury that is associated with anupregulation of IL-11, e.g. an upregulation of IL-11 in cells or tissuein which the symptoms of the disease/disorder/condition manifests or mayoccur, or upregulation of extracellular IL-11 or IL-11Rα.

The disorder, disease or condition associated with kidney injury mayaffect any tissue or organ or organ system. In some embodiments, thedisease/disorder/condition may affect several tissues/organs/organsystems. In some embodiments, the disease/disorder/condition affects thekidney.

In some embodiments, the disorder, disease or condition associated withkidney injury affects one or more of: the cardiovascular system, thedigestive system, the excretory system, the respiratory system, therenal system, the reproductive system, the circulatory system, themuscular system, the endocrine system, the exocrine system, thelymphatic system, the immune system, the nervous system, and/or theskeletal system.

In some embodiments, the present invention provides for thetreatment/prevention of kidney injury-related pathology in acute kidneyinjury, nephrotoxicity, drug-induced kidney injury, drug-induced acutekidney injury, drug-induced nephrotoxicity, cisplatin-induced kidneyinjury, cisplatin-induced acute kidney injury, cisplatin-inducednephrotoxicity.

Treatment may be effective to prevent progression of kidney injuryand/or a disorder, disease or condition associated with kidney injury,e.g. to reduce/delay/prevent worsening of, or to reduce/delay/preventdevelopment of, kidney injury and/or a disorder, disease or conditionassociated with kidney injury. In some embodiments treatment may lead toan improvement, e.g. a reduction in the severity of, and/or a reversalof, the symptoms of kidney injury and/or a disorder, disease orcondition associated with kidney injury. In some embodiments treatmentmay increase survival. In some embodiments treatment is effective toreverse the effects and/or symptoms of kidney injury and/or a disorder,disease or condition associated with kidney injury.

Prevention may refer to prevention of development of kidney injuryand/or a disorder, disease or condition associated with kidney injury,and/or prevention of worsening of kidney injury and/or a disorder,disease or condition associated with kidney injury, e.g. prevention ofprogression of kidney injury and/or a disorder, disease or conditionassociated with kidney injury to a later or chronic stage.

In some embodiments, the present invention provides for thetreatment/prevention of acute kidney injury (AKI), acute kidney failure,acute kidney disease, chronic kidney disease, kidney damage,drug-induced kidney injury, tubular necrosis, acute tubular necrosis,autoimmune kidney injury and cancer.

Acute tubular necrosis is the most common type of intrinsic acute kidneyinjury in hospitalized patients (Rahman M et al supra). The cause isoften ischemic (from prolonged hypotension) or nephrotoxic (from anagent that is toxic to the tubular cells). Acute kidney injury caused byacute tubular necrosis often does not improve with adequate repletion ofintravascular volume and blood flow to the kidneys. Both ischemic andnephrotoxic acute tubular necrosis can resolve over time, althoughtemporary renal replacement therapy may be required, depending on thedegree of renal injury and the presence of preexisting chronic kidneydisease.

A “cancer” as referred to herein may be any unwanted cell proliferation(or any disease manifesting itself by unwanted cell proliferation),neoplasm or tumour or increased risk of or predisposition to theunwanted cell proliferation, neoplasm or tumour. The cancer may bebenign or malignant and may be primary or secondary (metastatic). Aneoplasm or tumour may be any abnormal growth or proliferation of cellsand may be located in any tissue. Examples of tissues include theadrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone,bone marrow, brain, breast, cecum, central nervous system (including orexcluding the brain) cerebellum, cervix, colon, duodenum, endometrium,epithelial cells (e.g. renal epithelia), gallbladder, oesophagus, glialcells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver,lung, lymph, lymph node (including abdominal lymph node, axillary lymphnode, cervical lymph node, inguinal lymph node, mediastinal lymph node,pelvic lymph node, periaortic lymph node), lymphoblast, maxilla,mediastinum, mesentery, myometrium, nasopharynx, omentume, oral cavity,ovary, pancreas, parotid gland, peripheral nervous system peritoneum,pleura, prostate, salivary gland, sigmoid colon, skin, small intestine,soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue,tonsil, trachea, uterus, vulva, white blood cells.

Cancers may be of a particular type. Examples of types of cancer includeastrocytoma, carcinoma (e.g. adenocarcinoma, hepatocellular carcinoma,medullary carcinoma, papillary carcinoma, squamous cell carcinoma),glioma, lymphoma, medulloblastoma, melanoma, myeloma, meningioma,neuroblastoma, sarcoma (e.g. angiosarcoma, chrondrosarcoma,osteosarcoma).

A “cancer” as used herein can comprise any one or more of the following:acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML),adrenocortical cancer, anal cancer, bladder cancer, blood cancer, bonecancer, brain tumor, breast cancer, cancer of the female genital system,cancer of the male genital system, central nervous system lymphoma,cervical cancer, childhood rhabdomyosarcoma, childhood sarcoma, chroniclymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colon andrectal cancer, colon cancer, endometrial cancer, endometrial sarcoma,esophageal cancer, eye cancer, gallbladder cancer, gastric cancer,gastrointestinal tract cancer, hairy cell leukemia, head and neckcancer, hepatocellular cancer, Hodgkin's disease, hypopharyngeal cancer,Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, leukemia,liver cancer, lung cancer, malignant fibrous histiocytoma, malignantthymoma, melanoma, mesothelioma, multiple myeloma, myeloma, nasal cavityand paranasal sinus cancer, nasopharyngeal cancer, nervous systemcancer, neuroblastoma, non-Hodgkin's lymphoma, oral cavity cancer,oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor,plasma cell neoplasm, primary CNS lymphoma, prostate cancer, rectalcancer, respiratory system, retinoblastoma, salivary gland cancer, skincancer, small intestine cancer, soft tissue sarcoma, stomach cancer,stomach cancer, testicular cancer, thyroid cancer, urinary systemcancer, uterine sarcoma, vaginal cancer, vascular system, Waldenstrom'smacroglobulinemia and Wilms' tumor.

In accordance with various aspects of the present invention, the methodsmay comprise one or more of the following (e.g. in the context of kidneyinjury):

-   -   Reducing necrosis of renal tissue and/or renal cells;    -   Reducing fibrosis of the kidney and/or renal tissue;    -   Reducing collagen content of, and/or inhibiting collagen        deposition in, the kidney and/or renal    -   tissue;    -   Increasing/maintaining renal function;    -   Increasing/maintaining urine output;    -   Reducing urinary albumin/creatinine ratio;    -   Reducing serum creatinine level;    -   Reducing serum urea level;    -   Reducing serum TGFβ1 level;    -   Increasing/maintaining kidney weight;    -   Increasing/maintaining renal cortical volume;    -   Increasing/maintaining body weight;    -   Inhibiting epithelial-to-mesenchymal cell transition of tubule        epithelial cells;    -   Reducing the number/proportion of ACTA2+ve cells in the kidney;    -   Reducing SNAIL expression by renal cells and/or in the        kidney/renal tissue; and    -   Increasing/maintaining E-cadherin expression by renal cells        and/or in the kidney/renal tissue.

Administration

Administration of an agent capable of inhibiting IL-11-mediatedsignalling is preferably in a “therapeutically effective” or“prophylactically effective” amount, this being sufficient to showbenefit to the subject.

In some embodiments, the agent may be administered before, inconjunction with, or after the cause of the kidney injury, e.g.administration or consumption of a nephrotoxic medicine or exposure toan environmental source of kidney injury.

The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of the kidneyinjury and the nature of the agent. Prescription of treatment, e.g.decisions on dosage etc., is within the responsibility of generalpractitioners and other medical doctors, and typically takes account ofthe disease/condition to be treated, the condition of the individualsubject, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of the techniques and protocolsmentioned above can be found in Remington's Pharmaceutical Sciences,20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

Multiple doses of the agent may be provided. One or more, or each, ofthe doses may be accompanied by simultaneous or sequentialadministration of another therapeutic agent.

Multiple doses may be separated by a predetermined time interval, whichmay be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or31 days, or 1, 2, 3, 4, 5, or 6 months. By way of example, doses may begiven once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).

In therapeutic applications, agents capable of inhibiting IL-11-mediatedsignalling are preferably formulated as a medicament or pharmaceuticaltogether with one or more other pharmaceutically acceptable ingredientswell known to those skilled in the art, including, but not limited to,pharmaceutically acceptable carriers, adjuvants, excipients, diluents,fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers,solubilisers, surfactants (e.g., wetting agents), masking agents,colouring agents, flavouring agents, and sweetening agents.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

Each carrier, adjuvant, excipient, etc. must also be “acceptable” in thesense of being compatible with the other ingredients of the formulation.

Suitable carriers, adjuvants, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbookof Pharmaceutical Excipients, 2nd edition, 1994.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theactive compound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with carriers(e.g., liquid carriers, finely divided solid carrier, etc.), and thenshaping the product, if necessary.

The formulations may be prepared for topical, parenteral, systemic,intravenous, intra-arterial, intramuscular, intrathecal, intraocular,intra-conjunctival, subcutaneous, oral or transdermal routes ofadministration which may include injection. Injectable formulations maycomprise the selected agent in a sterile or isotonic medium. Theformulation and mode of administration may be selected according to theagent and disease/disorder/condition to be treated.

In some cases, an article (e.g. agent/composition) as described hereinis administered for treatment as described herein in conjunction withtreatment for a disease/disorder/condition associated with kidneyinjury. Suitable treatments for a disease/disorder/condition associatedwith kidney injury are known in the art. A composition may beadministered alone or in combination with other treatments, eithersimultaneously or sequentially dependent upon thedisease/disorder/condition to be treated. For example, the article maybe administered before, at the same time as, or after the treatment. Thearticle and the treatment may be formulated together, e.g. in aformulation described above, or formulated separately.

Detection of IL-11 and Receptors for IL-11

Some aspects and embodiments of the present invention concern detectionof expression of IL-11 or a receptor for IL-11 (e.g. IL-11Rα, gp130, ora complex containing IL-11Rα and/or gp130) in a sample obtained from asubject.

In some aspects and embodiments the present invention concerns theupregulation of expression (over-expression) of IL-11 or a receptor forIL-11 (as a protein or oligonucleotide encoding the respective IL-11 orreceptor for IL-11) and detection of such upregulation as an indicatorof suitability for treatment with an agent capable of inhibiting theaction of IL-11 or with an agent capable of preventing or reducing theexpression of IL-11 or a receptor for IL-11.

Upregulated expression comprises expression at a level that is greaterthan would normally be expected for a cell or tissue of a given type.Upregulation may be determined by measuring the level of expression ofthe relevant factor in a cell or tissue. Comparison may be made betweenthe level of expression in a cell or tissue sample from a subject and areference level of expression for the relevant factor, e.g. a value orrange of values representing a normal level of expression of therelevant factor for the same or corresponding cell or tissue type. Insome embodiments reference levels may be determined by detectingexpression of IL-11 or a receptor for IL-11 in a control sample, e.g. incorresponding cells or tissue from a healthy subject or from healthytissue of the same subject. In some embodiments reference levels may beobtained from a standard curve or data set.

Levels of expression may be quantitated for absolute comparison, orrelative comparisons may be made.

In some embodiments upregulation of IL-11 or a receptor for IL-11 (e.g.IL-11Rα, gp130, or a complex containing IL-11Rα and/or gp130) may beconsidered to be present when the level of expression in the test sampleis at least 1.1 times that of a reference level. More preferably, thelevel of expression may be selected from one of at least 1.2, at least1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least2.3, at least 2.4 at least 2.5, at least 2.6, at least 2.7, at least2.8, at least 2.9, at least 3.0, at least 3.5, at least 4.0, at least5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, or at least10.0 times that of the reference level.

Expression levels may be determined by one of a number of known in vitroassay techniques, such as PCR based assays, in situ hybridisationassays, flow cytometry assays, immunological or immunohistochemicalassays.

By way of example suitable techniques involve a method of detecting thelevel of IL-11 or a receptor for IL-11 in a sample by contacting thesample with an agent capable of binding IL-11 or a receptor for IL-11and detecting the formation of a complex of the agent and IL-11 orreceptor for IL-11. The agent may be any suitable binding molecule, e.g.an antibody, polypeptide, peptide, oligonucleotide, aptamer or smallmolecule, and may optionally be labelled to permit detection, e.g.visualisation, of the complexes formed.

Suitable labels and means for their detection are well known to those inthe art and include fluorescent labels (e.g. fluorescein, rhodamine,eosine and NDB, green fluorescent protein (GFP), chelates of rare earthssuch as europium (Eu), terbium (Tb) and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin,Cy3, Cy5), isotope markers, radioisotopes (e.g. 32P, 33P, 35S),chemiluminescence labels (e.g. acridinium ester, luminol, isoluminol),enzymes (e.g. peroxidase, alkaline phosphatase, glucose oxidase,beta-galactosidase, luciferase), antibodies, ligands and receptors.Detection techniques are well known to those of skill in the art and canbe selected to correspond with the labelling agent. Suitable techniquesinclude PCR amplification of oligonucleotide tags, mass spectrometry,detection of fluorescence or colour, e.g. upon enzymatic conversion of asubstrate by a reporter protein, or detection of radioactivity.

Assays may be configured to quantify the amount of IL-11 or receptor forIL-11 in a sample. Quantified amounts of IL-11 or receptor for IL-11from a test sample may be compared with reference values, and thecomparison used to determine whether the test sample contains an amountof IL-11 or receptor for IL-11 that is higher or lower than that of thereference value to a selected degree of statistical significance.

Quantification of detected IL-11 or receptor for IL-11 may be used todetermine up- or down-regulation or amplification of genes encodingIL-11 or a receptor for IL-11. In cases where the test sample containsfibrotic cells, such up-regulation, down-regulation or amplification maybe compared to a reference value to determine whether any statisticallysignificant difference is present.

A sample obtained from a subject may be of any kind. A biological samplemay be taken from any tissue or bodily fluid, e.g. a blood sample,blood-derived sample, serum sample, lymph sample, semen sample, salivasample, synovial fluid sample. A blood-derived sample may be a selectedfraction of a patient's blood, e.g. a selected cell-containing fractionor a plasma or serum fraction. A sample may comprise a tissue sample orbiopsy; or cells isolated from a subject. Samples may be collected byknown techniques, such as biopsy or needle aspirate. Samples may bestored and/or processed for subsequent determination of IL-11 expressionlevels.

Samples may be used to determine the upregulation of IL-11 or receptorfor IL-11 in the subject from which the sample was taken.

In some preferred embodiments a sample may be a tissue sample, e.g.biopsy, taken from kidney tissue, cardiac tissue, visceral organ tissue,respiratory system organ tissue, or urinary/renal system tissue. Asample may contain cells.

A subject may be selected for therapy/prophylaxis in accordance with thepresent invention based on determination that the subject has anupregulated level of expression of IL-11 or of a receptor for IL-11(e.g. IL-11Rα, gp130, or a complex containing IL-11R and/or gp130).Upregulated expression of IL-11 or of a receptor for IL-11 may serve asa marker of kidney injury and/or a disorder, disease or conditionassociated with kidney injury suitable for treatment with an agentcapable of inhibiting IL-11 mediated signalling.

Upregulation may be in a given tissue or in selected cells from a giventissue. A preferred tissue may be kidney/renal tissue. Upregulation ofexpression of IL-11 or of a receptor for IL-11 may also be determined ina circulating fluid, e.g. blood, or in a blood derived sample.Upregulation may be of extracellular IL-11 or IL-11Rα. In someembodiments expression may be locally or systemically upregulated.

Following selection, a subject may be administered with an agent capableof inhibiting IL-11 mediated signalling.

Diagnosis and Prognosis

Detection of upregulation of expression of IL-11 or a receptor for IL-11(e.g. IL-11Rα, gp130, or a complex containing IL-11Rα and/or gp130) mayalso be used in a method of diagnosing kidney injury and/or a disorder,disease or condition associated with kidney injury, identifying asubject at risk of developing kidney injury and/or a disorder, diseaseor condition associated with kidney injury, and in methods of prognosingor predicting a subject's response to treatment with an agent capable ofinhibiting IL-11 mediated signalling.

“Developing”, “development” and other forms of “develop” may refer tothe onset of a disorder/disease, or the continuation or progression of adisorder/disease.

In some embodiments a subject may be suspected of having or sufferingfrom kidney injury and/or a disorder, disease or condition associatedwith kidney injury, e.g. based on the presence of other symptomsindicative of kidney injury and/or a disorder, disease or conditionassociated with kidney injury in the subject's body or in selectedcells/tissues of the subject's body, or be considered at risk ofdeveloping kidney injury and/or a disorder, disease or conditionassociated with kidney injury, e.g. because of genetic predisposition orexposure to environmental conditions, known to be risk factors forkidney injury and/or a disorder, disease or condition associated withkidney injury. Determination of upregulation of expression of IL-11 or areceptor for IL-11 may confirm a diagnosis or suspected diagnosis, ormay confirm that the subject is at risk of developing kidney injuryand/or a disorder, disease or condition associated with kidney injury.The determination may also diagnose kidney injury and/or a disorder,disease or condition associated with kidney injury or predisposition asone suitable for treatment with an agent capable of inhibitingIL-11-mediated signalling.

As such, a method of providing a prognosis for a subject having, orsuspected of having kidney injury and/or a disorder, disease orcondition associated with kidney injury may be provided, the methodcomprising determining whether the expression of IL-11 or a receptor forIL-11 is upregulated in a sample obtained from the subject and, based onthe determination, providing a prognosis for treatment of the subjectwith an agent capable of inhibiting IL-11-mediated signalling.

In some aspects, methods of diagnosis or methods of prognosing orpredicting a subject's response to treatment with an agent capable ofinhibiting IL-11-mediated signalling may not require determination ofthe expression of IL-11 or a receptor for IL-11, but may be based ondetermining genetic factors in the subject that are predictive ofupregulation of expression or activity. Such genetic factors may includethe determination of genetic mutations, single nucleotide polymorphisms(SNPs) or gene amplification in IL-11, IL-11Rα and/or gp130 which arecorrelated with and/or predictive of upregulation of expression oractivity and/or IL-11 mediated signalling. The use of genetic factors topredict predisposition to a disease state or response to treatment isknown in the art, e.g. see Peter Stärkel Gut 2008; 57:440-442; Wright etal., Mol. Cell. Biol. March 2010 vol. 30 no. 6 1411-1420.

Genetic factors may be assayed by methods known to those of ordinaryskill in the art, including PCR based assays, e.g. quantitative PCR,competitive PCR. By determining the presence of genetic factors, e.g. ina sample obtained from a subject, a diagnosis may be confirmed, and/or asubject may be classified as being at risk of developing kidney injuryand/or a disorder, disease or condition associated with kidney injury,and/or a subject may be identified as being suitable for treatment withan agent capable of inhibiting IL-11 mediated signalling.

Some methods may comprise determination of the presence of one or moreSNPs linked to secretion of IL-11 or susceptibility to development ofkidney injury and/or a disorder, disease or condition associated withkidney injury. SNPs are usually bi-allelic and therefore can be readilydetermined using one of a number of conventional assays known to thoseof skill in the art (e.g. see Anthony J. Brookes. The essence of SNPs.Gene Volume 234, Issue 2, 8 Jul. 1999, 177-186; Fan et al., HighlyParallel SNP Genotyping. Cold Spring Harb Symp Quant Biol 2003. 68:69-78; Matsuzaki et al., Parallel Genotyping of Over 10,000 SNPs using aone-primer assay on a high-density oligonucleotide array. Genome Res.2004. 14: 414-425).

The methods may comprise determining which SNP allele is present in asample obtained from a subject. In some embodiments determining thepresence of the minor allele may be associated with increased IL-11secretion or susceptibility to development of kidney injury and/or adisorder, disease or condition associated with kidney injury.

Accordingly, in one aspect of the present invention a method forscreening a subject is provided, the method comprising:

-   -   obtaining a nucleic acid sample from the subject;    -   determining which allele is present in the sample at the        polymorphic nucleotide position of one or more of the SNPs        listed in FIG. 33, FIG. 34, or FIG. 35 of WO 2017/103108 A1        (incorporated by reference herein), or a SNP in linkage        disequilibrium with one of the listed SNPs with an r²≥0.8.

The determining step may comprise determining whether the minor alleleis present in the sample at the selected polymorphic nucleotideposition. It may comprise determining whether 0, 1 or 2 minor allelesare present.

The screening method may be, or form part of, a method for determiningsusceptibility of the subject to development of kidney injury and/or adisorder, disease or condition associated with kidney injury, or amethod of diagnosis or prognosis as described herein.

The method may further comprise the step of identifying the subject ashaving susceptibility to, or an increased risk of, developing kidneyinjury and/or a disorder, disease or condition associated with kidneyinjury, e.g. if the subject is determined to have a minor allele at thepolymorphic nucleotide position. The method may further comprise thestep of selecting the subject for treatment with an agent capable ofinhibiting IL-11 mediated signalling and/or administering an agentcapable of inhibiting IL-11 mediated signalling to the subject in orderto provide a treatment for kidney injury and/or a disorder, disease orcondition associated with kidney injury in the subject or to preventdevelopment or progression of kidney injury and/or a disorder, diseaseor condition associated with kidney injury in the subject.

In some embodiments, a method of diagnosing kidney injury and/or adisorder, disease or condition associated with kidney injury,identifying a subject at risk of developing kidney injury and/or adisorder, disease or condition associated with kidney injury, andmethods of prognosing or predicting a subject's response to treatmentwith an agent capable of inhibiting IL-11 mediated signalling employs anindicator that is not detection of upregulation of expression of IL-11or a receptor for IL-11, or genetic factors.

In some embodiments, a method of diagnosing kidney injury and/or adisorder, disease or condition associated with kidney injury,identifying a subject at risk of developing kidney injury and/or adisorder, disease or condition associated with kidney injury, andmethods of prognosing or predicting a subject's response to treatmentwith an agent capable of inhibiting IL-11 mediated signalling is basedon detecting, measuring and/or identifying one or more of the followingindicators or performing one of the following analyses:

-   -   Elevated serum creatinine, blood urea and/or nitrogen    -   Urinary sodium and creatinine levels    -   Reduced urine output    -   Renal ultrasonography to identify presence/absence of tubular        obstruction    -   Renal biopsy    -   Performing urinalysis.

Reference levels for laboratory kidney tests can be found in e.g. Rahmanet al supra, which is hereby incorporated by reference in its entirety.

Methods of diagnosis or prognosis may be performed in vitro on a sampleobtained from a subject, or following processing of a sample obtainedfrom a subject. Once the sample is collected, the patient is notrequired to be present for the in vitro method of diagnosis or prognosisto be performed and therefore the method may be one which is notpractised on the human or animal body. The sample obtained from asubject may be of any kind, as described herein above.

Other diagnostic or prognostic tests may be used in conjunction withthose described here to enhance the accuracy of the diagnosis orprognosis or to confirm a result obtained by using the tests describedhere.

Subjects

Subjects may be animal or human. Subjects are preferably mammalian, morepreferably human. The subject may be a non-human mammal, but is morepreferably human. The subject may be male or female. The subject may bea patient. The patient may have kidney injury and/or a disorder, diseaseor condition associated with kidney injury as described herein. Asubject may have been diagnosed with kidney injury and/or a disorder,disease or condition associated with kidney injury requiring treatment,may be suspected of having such kidney injury and/or a disorder, diseaseor condition associated with kidney injury, or may be at risk fromdeveloping kidney injury and/or a disorder, disease or conditionassociated with kidney injury.

In embodiments according to the present invention the subject ispreferably a human subject. In embodiments according to the presentinvention, a subject may be selected for treatment according to themethods based on characterisation for certain markers of kidney injuryand/or a disorder, disease or condition associated with kidney injury.

In some embodiments the subject may be a subject who is beingadministered a drug or medicine in order to treat a disease, conditionor disorder that may or may not be manifest in kidney tissue. Thesubject may have developed a drug-induced kidney injury as aconsequence, e.g. side-effect, of such treatment. The subject may beselected for treatment or preventative therapy according to thisinvention on the basis of having developed such drug-induced kidneyinjury.

In some embodiments, the subject may have received treatment with achemotherapeutic agent, or may be receiving treatment with achemotherapeutic agent. The subject may have, be suspected of having, orbe in recovery or remission from, cancer and administration of thechemotherapeutic agent may form part of the subject's treatment.Accordingly, the subject may be a subject having chemotherapeuticagent-induced kidney injury, chemotherapeutic agent-induced acute kidneyinjury or chemotherapeutic agent-induced nephrotoxicity.

In some preferred embodiments, the subject may have received treatmentwith cisplatin, or may be receiving treatment with cisplatin. Thesubject may have, be suspected of having, or be in recovery or remissionfrom, cancer and administration of cisplatin may form part of thesubject's treatment. Accordingly, the subject may be a subject havingcisplatin-induced kidney injury, cisplatin-induced acute kidney injuryor cisplatin-induced nephrotoxicity.

A subject may optionally be receiving intermittent or regular dialysis.

Sequence Identity

Pairwise and multiple sequence alignment for the purposes of determiningpercent identity between two or more amino acid or nucleic acidsequences can be achieved in various ways known to a person of skill inthe art, for instance, using publicly available computer software suchas ClustalOmega (Söding, J. 2005, Bioinformatics 21, 951-960), T-coffee(Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign(Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT(Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780software. When using such software, the default parameters, e.g. for gappenalty and extension penalty, are preferably used.

Sequences SEQ ID NO: DESCRIPTION SEQUENCE  1 Human IL-11 (UniProtMNCVCRLVLVVLSLWPDTAVAPGPPPGPPRVSPDPRAELDSTVLLTRSLLADTRQLA P20809)AQLRDKFPADGDHNLDSLPTLAMSAGALGALQLPGVLTRLRADLLSYLRHVQWLRRAGGSSLKTLEPELGTLQARLDRLLRRLQLLMSRLALPQPPPDPPAPPLAPPSSAWGGIRAAHAILGGLHLTLDWAVRGLLLLKTRL  2 Human gp130MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDY(UniProt P40189-1)HFVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQEDGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQ GGYMPQ  3Human IL11 RA MSSSCSGLSRVLVAVATALVSASSPCPQAWGPPGVQYGQPGRSVKLCCPGVTAGDPV(UniProt Q14626)SWFRDGEPKLLQGPDSGLGHELVLAQADSTDEGTYICQTLDGALGGTVTLQLGYPPARPVVSCQAADYENFSCTWSPSQISGLPTRYLTSYRKKTVLGADSQRRSPSTGPWPCPQDPLGAARCWHGAEFWSQYRINVTEVNPLGASTRLLDVSLQSILRPDPPQGLRVESVPGYPRRLRASWTYPASWPCQPHFLLKFRLQYRPAQHPAWSTVEPAGLEEVITDAVAGLPHAVRVSARDFLDAGTWSTWSPEAWGTPSTGTIPKEIPAWGQLHTQPEVEPQVDSPAPPRPSLQPHPRLLDHRDSVEQVAVLASLGILSFLGLVAGALALGLWLRLRRGGKDGSPKPGFLASVIPVDRRPGAPNL  4 siRNA target IL-11 CCTTCCAAAGCCAGATCTT  5siRNA target IL-11 GCCTGGGCAGGAACATATA  6 siRNA target IL-11CCTGGGCAGGAACATATAT  7 siRNA target IL-11 GGTTCATTATGGCTGTGTT  8siRNA target IL-11 Ra GGACCATACCAAAGGAGAT  9 siRNA target IL-11 RaGCGTCTTTGGGAATCCTTT 10 siRNA target IL-11 Ra GCAGGACAGTAGATCCCT 11siRNA target IL-11 Ra GCTCAAGGAACGTGTGTAA 12 siRNA to IL-11CCUUCCAAAGCCAGAUCUUdTdT-AAGAUCUGGCUUUGGAAGGdTdT (NM_000641.3) 13siRNA to IL-11 GCCUGGGCAGGAACAUAUAdTdT-UAUAUGUUCCUGCCCAGGCdTdT(NM_000641.3) 14 siRNA to IL-11CCUGGGCAGGAACAUAUAUdTdT-AUAUAUGUUCCUGCCCAGGdTdT (NM_000641.3) 15siRNA to IL-11 GGUUCAUUAUGGCUGUGUUdTdT-AACACAGCCAUAAUGAACCdTdT(NM_000641.3) 16 siRNA to IL-11 RaGGACCAUACCAAAGGAGAUdTdT-AUCUCCUUUGGUAUGGUCCdTdT (U32324.1) 17siRNA to IL-1 1 Ra GCGUCUUUGGGAAUCCUUUdTdT-AAAGGAUUCCCAAAGACGCdTdT(U32324.1) 18 siRNA to IL-11 RaGCAGGACAGUAGAUCCCUAdTdT-UAGGGAUCUACUGUCCUGCdTdT (U32324.1) 19siRNA to IL-11 Ra GCUCAAGGAACGUGUGUAAdTdT-UUACACACGUUCCUUGAGCdTdT(U32324.1) 20 20 amino acid linker GPAGQSGGGGGSGGGSGGGSV 21Hyper IL-11 (IL-11RA:MSSSCSGLSRVLVAVATALVSASSPCPQAWGPPGVQYGQPGRSVKLCCPGVTAGDPV IL-11 fusion)SWFRDGEPKLLQGPDSGLGHELVLAQADSTDEGTYICQTLDGALGGTVTLQLGYPPARPVVSCQAADYENFSCTWSPSQISGLPTRYLTSYRKKTVLGADSQRRSPSTGPWPCPQDPLGAARCVVHGAEFWSQYRINVTEVNPLGASTRLLDVSLQSILRPDPPQGLRVESVPGYPRRLRASWTYPASWPCQPHFLLKFRLQYRPAQHPAWSTVEPAGLEEVITDAVAGLPHAVRVSARDFLDAGTWSTWSPEAWGTPSTGPAGQSGGGGGSGGGSGGGSVPGPPPGPPRVSPDPRAELDSTVLLTRSLLADTRQLAAQLRDKFPADGDHNLDSLPTLAMSAGALGALQLPGVLTRLRADLLSYLRHVQWLRRAGGSSLKTLEPELGTLQARLDRLLRRLQLLMSRLALPQPPPDPPAPPLAPPSSAWGGIRAAHAILGGLHLTLDWAVRGLLLLK TRL 22Enx203 VH EVQLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPHNGGPIYNQKFTGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARGELGHWYFDVWGTGTT VTVSS 23Enx203 VL DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYIHWYQQKPGQPPKLLIYLASNLDSGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRDLPPTFGGGTKLEIK 24 Enx209 VHQVQLQQPGAELVRPGSSVKLSCKASGYTFTNYWMHWLKQRPVQGLEWIGNIGPSDSKTHYNQKFKDKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARGDYVLFTYWGQGTLVT VSA 25Enx209 VL DIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKSHEAPRLLIYASQSISGIPARFSGSGSGTDFTLSFSSLETEDFAVYFCQQSYSWPLTFGQGTKLEIK 26 Enx108A VHQVQLVQSGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIGATDPLDYWGQGTLVT VSS 27Enx108.A VL QSALTQPRSVSGSPGQSVTLSCTGTSSDVGGYNYVSWYQHYPGKAPKLMIFDVNERSSGVPDRFSGSKSGNTASLTiSGLQAEDEADYYCASYAGRYTWMFGGGTKVTVLG 28 EnxA hlgG4QVQLVQSGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNK(L248E, S241P) HCYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIGATDPLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMiSRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 29 Enx108A lambda LCQSALTQPRSVSGSPGQSVTLSCTGTSSDVGGYNYVSWYQHYPGKAPKLMIFDVNERSSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCASYAGRYTWMFGGGTKVTVLGQPKAAPSVILFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 30 hEnx203 VHEVQLVQSGAEVKKPGASVKISCKASGYTFTDYNMDWVKQAPGQRLEWIGDINPHNGGPIYNQKFTGRATLTVDKSASTAYMELSSLRSEDTAVYYCARGELGHWYFDVWGQGTT VTVSS 31hEnx203 VL DIVLTQSPASLALSPGERATLSCRASKSVSTSGYSYIHWYQQKPGQAPRLLIYLASNLDSGVPARFSGSGSGTDFTLTISSLEEEDFATYYCQHSRDLPPTFGQGTKLEIK 32 hEnx209 VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWLRQRPGQGLEWIGNIGPSDSKTHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGDYVLFTYWGQGTLVT VSS 33hEnx209 VL DIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIKYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSYSWPLTFGQGTKLEIK 34 Enx108A VH CDR1SYGMH 35 Enx108A VH CDR2 VISYDGSNKYYADSVKG 36 Enx108A VH CDR3 IGATDPLDY37 Enx108A VL CDR1 TGTSSDVGGYNYVS 38 Enx108A VL CDR2 DVNERSS 39Enx108A VL CDR3 ASYAGRYTWM 40 Enx203, hEnx203 VH DYNMD CDR1 41Enx203, hEnx203 VH DINPHNGGPIYNQKFTG CDR2 42 Enx203, hEnx203 VHGELGHWYFDV CDR3 43 Enx203, hEnx203 VL RASKSVSTSGYSYIH CDR1 44Enx203, hEnx203 VL LASNLDS CDR2 45 Enx203, hEnx203 VL QHSRDLPPT CDR3 46Enx209, hEnx209 VH NW MH CDR1 47 Enx209, hEnx209 VH NIGPSDSKTHYNQKFKDCDR2 48 Enx209, hEnx209 VH GDYVLFTY CDR3 49 Enx209, hEnx209 VLRASQSISNNLH CDR1 50 Enx209, hEnx209 VL YASQSIS CDR2 51Enx209, hEnx209 VL QQSYSWPLT CDR3 52 Human IGHG1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLconstant (K214R,QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA D356E, L358M)PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 53 Human IGHG4 constantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL (L248E, S241P)QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 54 Human IGKCRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT constantEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 55 Human IGLC2GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTT constantPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 56 hEnx203 hlgG1 HCEVQLVQSGAEVKKPGASVKISCKASGYTFTDYNMDWVKQAPGQRLEWIGDINPHNGGPIYNQKFTGRATLTVDKSASTAYMELSSLRSEDTAVYYCARGELGHWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 57 hEnx203 kappa LCDIVLTQSPASLALSPGERATLSCRASKSVSTSGYSYIHWYQQKPGQAPRLLIYLASNLDSGVPARFSGSGSGTDFTLTISSLEEEDFATYYCQHSRDLPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAGEVTHQGLSSPVTKSFNRGEC 58 hEnx209 hlgG4QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWLRQRPGQGLEWIGNIGPSDSK(L248E, S241P) HCTHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARGDYVLFTYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 59 hEnx209 kappa LCDIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIKYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSYSWPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The features disclosed in the foregoing description, or in the followingclaims, or in the accompanying drawings, expressed in their specificforms or in terms of a means for performing the disclosed function, or amethod or process for obtaining the disclosed results, as appropriate,may, separately, or in any combination of such features, be utilised forrealising the invention in diverse forms thereof.

For the avoidance of any doubt, any theoretical explanations providedherein are provided for the purposes of improving the understanding of areader. The inventors do not wish to be bound by any of thesetheoretical explanations.

Any section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise” and “include”, andvariations such as “comprises”, “comprising”, and “including” will beunderstood to imply the inclusion of a stated integer or step or groupof integers or steps but not the exclusion of any other integer or stepor group of integers or steps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment. The term “about” in relation to anumerical value is optional and means for example +/−10%.

Methods disclosed herein may be performed, or products may be present,in vitro, ex vivo, or in vivo. The term “in vitro” is intended toencompass experiments with materials, biological substances, cellsand/or tissues in laboratory conditions or in culture whereas the term“in vivo” is intended to encompass experiments and procedures withintact multi-cellular organisms. “Ex vivo” refers to something presentor taking place outside an organism, e.g. outside the human or animalbody, which may be on tissue (e.g. whole organs) or cells taken from theorganism.

Where a nucleic acid sequence is disclosed herein, the reversecomplement thereof is also expressly contemplated.

For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold SpringHarbor, New York: Cold Spring Harbor Laboratory Press Aspects andembodiments of the present invention will now be discussed withreference to the accompanying figures. Further aspects and embodimentswill be apparent to those skilled in the art. All documents mentioned inthis text are incorporated herein by reference. While the invention hasbeen described in conjunction with the exemplary embodiments describedbelow, many equivalent modifications and variations will be apparent tothose skilled in the art when given this disclosure. Accordingly, theexemplary embodiments of the invention set forth above are considered tobe illustrative and not limiting.

Various changes to the described embodiments may be made withoutdeparting from the spirit and scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures.

FIG. 1 . Micrographs showing neutralising anti-IL-11 antibodies preventprogression from AKI to CKD as compared to IgG control antibody. Micewere subjected to folate induced kidney injury (a model of acute tubularnecrosis (ATN) and secondary fibrosis) and administered either IgGcontrol, Enx203 (anti-IL-11 antibody) or ENx209 (anti-IL-11RA antibody).Kidneys were harvested after 28 days and used for 35 Masson's trichromestaining. Both antibodies were remarkably effective at preventingcollagen deposition.

FIGS. 2A and 2B. Charts showing neutralising anti-IL-11 antibodiesprevent AKI progression. Mice were subjected to folate induced kidneyinjury and administered either IgG control or anti-IL-11 antibody(Enx203, AB1) or anti-IL11RA antibody (ENx209, AB2). At 28 days, kidneyswere collected for HPA collagen assay and serum urine (collected inmetabolic cages) assessed for urinary albumin/creatinine ratios.Note—day 2 makers of kidney injury were similar between treatment groupsshowing equal levels of initial renal damage but mice receivinganti-IL-11 therapies largely recovered function whereas those receivingIgG less effectively recovered function. (2A) Shows renal collagencontent, and (2B) shows urinary albumin/creatinine ratios at day 28 formice subjected to different treatments.

FIG. 3 . Graph and image showing dose-dependent effects of ENx203 andENx209 on kidney injury responses and ERK activation. Animals weretreated with folate and antibodies were injected from one hour beforeinjury. Renal collagen content was assessed after 28 days. Animals weretreated with either 10 mg/kg or 20 mg/kg biweekly with IgG controlantibody. No difference in between groups was observed and animals fromboth groups are plotted together. The folate model of kidney injury isvery severe (see FIG. 4 ). However, even in the context of the strongchemical stimulus, ENx203 at doses down to 5 mg/kg twice a week washighly effective (upper panel). Individuals points indicate biologicalreplicates. Data is shown as mean±SD. Biweekly (biw.) and triweekly(triw.) injection of antibodies. Monitoring IL-11-mediated ERKactivation in the fibrotic kidney as a read out of target engagement(lower panel), the inventors were able to show effects of ExN203 at 1mg/kg twice a week.

FIGS. 4A to 4C. Graphs showing dose-dependent effects of ENx203 andENx209 on (4A) serum creatinine, (4B) serum urea and (4C) serum TGFB1levels. Animals were treated with folate and antibodies were injectedfrom one hour before injury. Serum marker levels were assessed after 28days. Animals were treated with either 10 mg/kg or 20 mg/kg biweeklywith IgG control antibody. No difference in between groups was observedand animals from both groups are plotted together. Individual pointsindicate biological replicates. Data is shown as mean±SD. Biweekly(biw.) and triweekly (triw.) injection of antibodies.

FIGS. 5A to 5C. Graphs showing the effects of treatment with (5A)ENx108A (IgG1), (5B) ENx203 or (5C) ENx209 on AKI progression. Mice weretreated with folate and from 1 day before were also treated withantibodies at given concentrations (biweekly). Renal collagen contentwas determined 21 days after kidney injury. Data points indicateindividual animals and all data was generated in parallel, so individualantibodies can be compared with each other. Identical baseline and IgGcontrol animals are plotted for each antibody to facilitate datainterpretation. Data are mean+−s.d.; Points indicate animals per group.Two-tailed Dunnett's test, corrected P-values.

FIGS. 6A and 6B. Graphs showing that fibroblast-specific knock out ofIl11ra in mice protects from AKI. Animals were treated with tamoxifen todelete Il1ra1 from Col1a1+ve cells prior to renal injury with folate.After 21 days (6A) collagen content was assessed using the HPA assay and(6B) kidney function was determined via serum urea levels. Animals wereprotected to similar levels as during antibody treatment, suggesting acentral role of IL-11 activity on fibroblasts in the pathogenesis ofrenal injury and dysfunction. Sidak corrected P-value.

FIGS. 7A to 7F. Schematic, graphs, and image relating to the effect ofanti-IL-11 therapy from day 3 post AKI. (7A) Schematic representation ofthe timing of folate administration and antibody treatment. (7B) Micereceiving anti-IL-11 therapy gain weight soon after receiving anti-IL-11therapy. (7C) At the end of the study period mice receiving anti-IL-11therapy had higher kidney weights, (7D) less renal collagen, (7E) normalurine outputs and (7F) improved gross morphology as compared to micereceiving IgG.

FIGS. 8A to 8F. Schematic, graphs, and image showing that ENx203treatment reverses kidney injury and dysfunction and induces kidneyregeneration. (8A) Schematic representation of the timing of folateadministration and antibody treatment. The folate model of kidney injurywas established, and animal were not treated the until 21 days afterinjury. This led to severe reduction of kidney function and increase incollagen content. Antibody treatment was then initiated, and analysis ofkidney function by evaluation of serum urea levels and collagen contentwas performed after 3, 6, 9 and 12 weeks of treatment. This revealed asignificant (8B) reduction in collagen content, and (8C) improvement inrenal function in ENx203-treated animals compared to animals before thetreatment at day 21. While kidneys contained less collagen, they still(8D) gained weight and (8E), (8F) looked less bumpy and had healthierhistology (cross-section of kidney, 3 animals). P values are correctedfor multiple testing and indicate differences between later time pointswith the FA 21d groups (Dunnett test). Data is shown as mean±SD.

FIG. 9 . Images showing that ENx203 treatment reduced collagen contentand promoted restoration of normal renal parenchyma and cortical volume.Masson's Trichrome stain of mid-section of kidneys from mice day 21(D21) after folic acid (FA) injury or control mice and then in micetreated from D21 post FA injury with either IgG or anti-IL-11 for 3, 6,9 or 12 weeks.

FIG. 10 . Images showing higher magnification view of the renal cortexfrom images shown in FIG. 9 . Masson's Trichrome stain of the cortex ofkidneys from mice day 21 (D21) after folic acid (FA) injury or controlmice and then in mice treated from D21 post FA injury with either IgG oranti-IL-11 for 3, 6, 9 or 12 weeks.

FIGS. 11A and 11B. Graphs showing that ENx203 treatment afterestablished renal injury induces weight gain and leads to a 50% reversalin urinary ACR. The folate model of kidney injury was performed, andanimals were not treated until 21 days after injury. Antibody treatmentwas then initiated, and animal weight was continually assessed. (11A)ENx203-treated animals started to regain weight upon initiation oftreatment. (11B) Urinary albumin:creatinine ratio was reversed by ˜50%by 12 weeks of therapy as compared to starting levels at 3-weeks postinjury.

FIGS. 12A and 12B. Graphs showing that TGFB1-driven partialepithelial-mesenchymal transformation of tubular epithelial cells isIL-11 dependent. Human primary tubule epithelial cells were stimulatedwith TGFB1, IL-11 (5 ng/ml, 24 h) or TGFB1 and antibody (2 μg/ml, 24 h).(12A) The percentage of ACTA2^(−ve) cells and (12B) collagen expressionwas monitored on the Operetta high-content imaging platform.

FIG. 13 . Images relating to TGFB1-driven partial epithelial-mesenchymaltransformation of tubular epithelial cells. Top, human primary tubuleepithelial cells were stimulated with IL-11 (2.5-20 ng/ml, 24 h).Operetta high-content imaging was used to visualise actin stress fibreformation using rhodamine-phalloidin. Bottom, visual representation ofcollagen expression in cells.

FIGS. 14A and 14B. Images and graph showing that expression of thefundamentally important Snail factor by Tubular epithelial cells (TECs)is IL-11 dependent. Primary human TECs were stimulated with either IL-11or TGFB in the presence of IgG or anti-IL-11 antibody (ENx203) andexpression of the Snail or ACTA2 gene detected using the Operettaimaging platform. (14A) both IL-11 and TGFB induce partial EMT andexpression of both ACTA2 and Snail. In the presence of anti-IL-11antibody this effect was strongly inhibited in TGFB stimulated cells.(14B) quantification of ACTA+ve cells from the micrographs of FIG. 14A.

FIG. 15 . Image showing that anti-IL-11 antibody treatment preventsSnail induction in primary human TECs. Western blot of primary humankidney tubular epithelial cells treated with TGFB in the presence orabsence of anti-IL-11 antibody (ENx203) or with IL-11. Upper panel,induction of SNAIL, the master regulator of EMT, by TGFB (5 ng/ml) isdependent on IL-11 signaling and IL-11 (5 ng/ml) alone is able to induceSNAIL expression. Lower panel, equal protein loading confirmed by GAPDHcontrol protein.

FIG. 16 . Image showing that E-cadherin expression in regeneratingkidneys is increased with anti-IL-11 therapy. E-cadherin expression isthe canonical marker of epithelial cell identity and expression ofE-cadherin is lost when cells undergo EMT or partial EMT. In keepingwith an effect of IL-11 driving TECs into a partial EMT (pEMT), Westernblot of kidneys from mice D21 post folate injury and those treated withIgG for 12 weeks after injury had lower E-cadherin levels compared tocontrols and this effect was reversed with anti-IL-11 therapy.

FIGS. 17A and 178 . Graph and bar chart relating to the effect ofanti-IL-11 antibody treatment in a Unilateral Ureter Obstruction (UUO)model of kidney injury. UUO was performed to induce physical renalinjury responses in mice. Some animals were treated with ENx203 or IgGcontrol antibody at day 4, 7 and 9 of treatment (20 mg/kg). (17A) Bodyweight of ENx203 and IgG control treated animals. (17B) Collagen contentin the kidney was assessed using the HPA assay 10 days after theprocedure. Animals treated with ENx203 therapeutic antibody hadsignificantly reduced collagen content compared to animals treated withcontrol antibody. Data is shown as mean±SD. Sidak corrected P-value.

FIGS. 18A to 18H. Schematic and graphs relating to the effect ofanti-IL-11 antibody treatment in a cisplatin-induced model of kidneyinjury. (18A) Mice were administered cisplatin (7 mg/kg cisplatin), onceweekly for 4 weeks. A control group was administered with saline. Micewere administered biweekly from week 1 by IP injection with 10 mg/kgX203 or an isotype-matched IgG control antibody. Mice were harvested foranalysis of the kidneys after 8 weeks. (18B) Collagen content in thekidney as assessed by the HPA assay. (18C to 18H) RNA expression of(18C) Col3, (18D) Fibronectin, (18E) MMP2, (18F) TIMP, (18G) CCL5 and(18H) CCL2 as determined by qPCR. Graphs show fold change (FC) inexpression relative to expression in saline-treated mice.

EXAMPLES

In the following examples, the inventors demonstrate that anti-IL-11therapy can ameliorate kidney injury through extensive regeneration andreversal of renal impairment in models of acute and chronic kidneydisease.

Example 1: Materials and Methods

1.1 Primary Human Renal Proximal Tubule Epithelial Cells

Culture and Stimulation Conditions

Primary human renal proximal tubule epithelial cells (HRPTEC,PCS-400-010, ATCC) were grown and maintained at 37° C. and 5% CO₂ incomplete renal epithelial cell growth medium (PCS-400-030 andPCS-400-040, ATCC). HRPTEC medium was renewed every 2-3 days and cellswere passaged at 80% confluence using standard trypsinizationtechniques. All the experiments were carried out at P3 and cells wereserum-starved for 16 hours prior to respective stimulations (24 hours)that were performed in serum-free renal epithelial cell media.Stimulated cells were compared to unstimulated cells that have beengrown for the same duration under the same conditions (serum-free renalepithelial cell media), but without the stimuli.

Operetta Phenotyping Assay

HRPTEC were seeded in 96-well CellCarrier plates (600550, PerkinElmer)at a density of 1×10⁴ cells per well. Cells were stimulated with theindicated concentrations of IL-11 (Genscript) or TGF-β1 (PHC 143B,Bio-Rad) with and without the presence of 2 μg/ml of either anti IL-11(ENx203) or IgG isotype control antibodies for 24 hours. Followingexperimental conditions, cells were fixed in 4% paraformaldehyde (PFA,28908, Thermo Fisher Scientilic), permeabilized with 0.1% Triton X-100(T8787, Sigma) in PBS. Non-specific binding sites were blocked 0.5% BSA(A7906, Sigma) and 0.1% Tween-20 (170-6531, Bio-Rad) in PBS. Cells wereincubated overnight (4 C) with primary antibodies (1:500) i.e. ACTA2(ab7817, Abcam), Collagen I (34710, Abcam), SNAIL (ab180714, Abcam) andfollowed by incubation with the appropriate AlexaFluor488-conjugatedsecondary antibodies (Goat anti-mouse 488, ab150113 and Goat anti-Rabbit488, ab150077, Abcam) for 1 hour (1:1000, RT, dark). Cells werecounter-stained with Rhodamine-Phalloidin (R415, Thermo FisherScientific) and DAPI (1 μg/ml, D1306, Thermo Fisher Scientific) inblocking solution. Plates were scanned and images were collected with anOperetta high-content imaging system (1483, PerkinElmer). Each conditionwas assayed from at least two wells and a minimum of seven fields perwell. The quantification of ACTA2+ cells was done using Harmony softwareversion 3.5.2. The measurement of Collagen I fluorescence intensity perarea was performed with Columbus version 2.7.1.

1.2 Western Blot

Western blot was carried out on HRPTE total protein extracts. HRPTE werelysed in radioimmunoprecipitation assay (RIPA) buffer containingprotease and phosphatase inhibitors (Thermo Scientifics), followed bycentrifugation to clear the lysate. Protein concentrations weredetermined by Bradford assay (Bio-Rad). Equal amount of protein lysateswere separated by SDS-PAGE, transferred to PVDF membrane, and subjectedto immunoblot analysis for SNAIL (3879, CST) and GAPDH (2118. CST).Proteins were visualized using the ECL detection system (Pierce) withanti-rabbit-HRP (7074, CST).

1.3 Animal Models

All animal procedures were approved and conducted in accordance with theSingHealth Institutional Animal Care and Use Committee (IACUC). All micewere provided food and water ad libitum.

Mouse Model of Chemically Induced-Acute Kidney Injury

Kidney injury was induced by IP injection of folic acid (200 mg/kg) invehicle (0.3M NaHCO₃) to 10 weeks old male mice; control mice wereadministered vehicle alone. Animals were sacrificed 28 days post-FA.Mice were intraperitoneally injected with anti-IL-11 antibody (ENx203),anti-IL-11Rα antibody (ENx209) or identical concentration of IgG isotypecontrol. Durations of treatment and dosage of antibody therapies areoutlined in the figures.

Mouse Model of Surgically Induced-Acute Kidney Injury

Unilateral ureteral obstruction (UUO) surgeries were carried out on 12weeks old male mice. Briefly, mice were anesthetized by IP injection ofketamine (100 mg/kg)/xylazine (10 mg/kg) and full depth of anaesthesiawas accessed with the pedal reflex. Mice were then shaved on the leftside of the abdomen. A vertical incision was made through the skin witha scalpel, a second incision was made through the peritoneum to revealthe kidney. Using forceps, the kidney was brought to the surface and theureter was tied with surgical silk, twice, below the kidney. The ligatedkidney was placed gently back into its correct anatomical position andsterile saline was added to replenish loss of fluid. The incisions werethen sutured. Animals were post-operatively treated with antibioticenrofloxacin (15 mg/kg, SC) and analgesic buprenorphine (0.1 mg/kg, SC)for three consecutive days.

1.4 Colorimetric Assays and ELISA

The levels of blood urea nitrogen (BUN) in mouse serum were measuredusing Urea Assay Kit (ab83362, Abcam). Urine albumin and creatininelevels were measured using Mouse Albumin ELISA kit (ab108792, Abcam) andCreatinine Assay Kit (ab204537, Abcam), respectively. Serum TGFβ1 levelswere measured by ELISA. All ELISA and colorimetric assays were performedaccording to the manufacturer's protocol.

1.5 Histology

Kidney tissues were fixed for 48 h at RT in 10% neutral-bufferedformalin (NBF), dehydrated, embedded in paraffin blocks and sectioned at4 μm. Sections were then stained with Masson's Trichrome according tostandard protocol and examined by light microscopy.

Example 2: Analysis of the Effect of Inhibition of IL-11 MediatedSignalling in a Model of Chemically-Induced Kidney Injury

Kidney injury was chemically-induced in 10-12 week old littermate miceof similar weight by intraperitoneal (i.p.) injection of folic acid (180mg kg⁻¹) in vehicle (0.3 M NaHCO₃); control mice were administeredvehicle alone.

Enx203 (an antibody capable of binding to mouse IL-11 (and human IL-11)and inhibiting IL-11 mediated signalling) or Enx209 (an antibody capableof binding to mouse IL-11 (and human IL-11) and inhibiting IL-11mediated signalling) were administered one day after folic acidtreatment, and then 3 times per week at a dose of 20 mg/kg. Mice wereeuthanized 28 days post-injection.

Enx203 and Enx209 are antagonists of IL-11 mediated signalling. Enx203is a mouse anti-mouse IL-11 IgG, and is described e.g. in Ng et al., SciTransl Med. (2019) 11(511) pii: eaaw1237 (also published as Ng, et al.,“IL-11 is a therapeutic target in idiopathic pulmonary fibrosis.”bioRxiv 336537; doi: https://doi.org/10.1101/336537). Enx203 is alsoreferred to as “X203”. Enx203 comprises the VH region according to SEQID NO:92 of WO 2019/238882 A1 (SEQ ID NO:22 of the present disclosure),and the VL region according to SEQ ID NO:94 of WO 2019/238882 A1 (SEQ IDNO:23 of the present disclosure). Enx209 is a mouse anti-mouse IL-11RαIgG, and is described e.g. in Widjaja et al., Gastroenterology (2019)157(3):777-792 (also published as Widjaja, et al., “IL-11 neutralisingtherapies target hepatic stellate cell-induced liver inflammation andfibrosis in NASH.” bioRxiv 470062; doi: https://doi.org/10.1101/470062).Enx209 is also referred to as “X209”. Enx209 comprises the VH regionaccording to SEQ ID NO:7 of WO 2019/238884 A1 (SEQ ID NO:24 of thepresent disclosure), and the VL region according to SEQ ID NO:14 of WO2019/238884 A1 (SEQ ID NO:25 of the present disclosure).

The mouse plasma levels of urea and creatinine were quantified usingurea assay kit (ab83362, Abcam) and creatinine assay kit (ab65340.Abcam), respectively according to the manufacturer's instructions. Theamount of total collagen in the kidney was quantified on the basis ofcolourimetric detection of hydroxyproline using a Quickzyme TotalCollagen assay kit (Quickzyme Biosciences). All colourimetric assayswere performed according to the manufacturer's instructions.

Tissues were paraffin-embedded, and kidneys were sectioned at 3 μm. Forparaffin sections, tissues were fixed for 24 h, at room temperature in10% neutral-buffered formalin (Sigma-Aldrich), dehydrated and embeddedin paraffin. For cryosections, freshly dissected organs were embeddedwith Tissue-Tek Optimal Cutting Temperature compound (VWRInternational). Cryomoulds were then frozen in a metal beaker withisopentane cooled in liquid nitrogen and sections were stored in −80° C.Total collagen was stained with Masson's trichrome stain kit (HT15,Sigma-Aldrich) according to the manufacturer's instructions. Images ofthe sections were captured and blue-stained areas weresemi-quantitatively determined with ImageJ software (version 1.49). Forimmunohistochemistry, the tissue sections were incubated with anti-ACTA2antibody (ab5694, Abcam). Primary antibody staining was visualized usingan ImmPRESS HRP Anti-Rabbit IgG Polymer Detection kit (VectorLaboratories) with ImmPACT DAB Peroxidase Substrate (VectorLaboratories) as the chromogen. The sections were then counterstainedwith Mayer's haematoxylin (Merck).

FIGS. 1 and 2 show that mice treated with anti-IL11 antibody oranti-IL-11Rα antibody antagonists were found to have significantlyreduced staining for collagen.

FIG. 2 also shows that the urinary albumin/creatine ratio wassignificantly reduced by treatment with anti-IL-11 antibody oranti-IL-11Rα antibody, indicating a reduced level of kidney damage.

Importantly, the serum levels of urea and creatine at day 2 followingchemical induction of kidney damage were similar between treatmentgroups (data not shown), however mice receiving antagonist IL-11therapies largely recovered function whereas those receiving IgG lesseffectively recovered function.

In a separate experiment, a similar folate-induced model of kidneyinjury was used to evaluate the therapeutic efficacy of antagonists ofIL-11 mediated signalling to treat/prevent kidney injury. Mice weretreated with folate as described above, and antibodies were administeredfrom one hour prior to injury. Renal collagen content was assessed after28 days. Animals were treated with 1 mg/kg Enx203 biweekly, 5 mg/kgEnx203 biweekly, 5 mg/kg Enx203 triweekly, 10 mg/kg Enx203 biweekly, 10mg/kg Enx203 triweekly, 20 mg/kg Enx203 triweekly, 20 mg/kg Enx209biweekly, or 10 mg/kg or 20 mg/kg IgG control antibody biweekly.

FIG. 3 shows that treatment with the antibody antagonists ofIL-11-mediated signalling reduced the levels of collagen contentassociated with folic acid-induced kidney injury in a dose-dependentfashion. An effect on collagen levels was seen even when Enx203 wasadministered at 5 mg/kg, biweekly.

Monitoring IL-11-mediated ERK activation in the fibrotic kidney as aread out of target engagement, the inventors demonstrated an effect forExN203 at a dose of 1 mg/kg, administered twice per week.

Serum levels of creatine, urea and TGFβ1 were also evaluated in theseexperiments, and FIG. 4 shows that treatment with the antibodyantagonists of IL-11-mediated signalling reduced serum levels of thesecorrelates of kidney injury in a dose-dependent manner. Again, reductionof the levels of these factors in serum was observed even when Enx203was administered at 5 mg/kg, biweekly.

In further experiments, a similar folate-induced model of kidney injurywas used to evaluate the therapeutic efficacy of antagonists of IL-11mediated signalling to treat/prevent kidney injury. Mice were treatedwith folate as described above, and antibodies were administered fromone day prior to injury. Renal collagen content was assessed after 21days. Animals were administered biweekly with 10 mg/kg IgG controlantibody, or 0.5 mg/kg, 1 mg/kg, 5 mg/kg or 10 mg/kg of Enx203, Enx209or Enx108A. The results are shown in FIG. 5 .

Enx108A is a human anti-human IL-11 IgG capable of binding to mouseIL-11 and human IL-11, and inhibiting IL-11 mediated signalling. Enx108Ais described e.g. in WO 2019/238882 A1, and comprises the VH regionaccording to SEQ ID NO:8 of WO 2019/238882 A1 (SEQ ID NO:26 of thepresent disclosure), and the VL region according to SEQ ID NO:20 of WO2019/238882 A1 (SEQ ID NO:27 of the present disclosure). In the presentExample. Enx108A is provided in hIgG4 (L248E, S241P), lambda lightformat (i.e. is formed of the heavy chain having the amino acid sequenceshown in SEQ ID NO:28, and the light chain having the amino acidsequence shown in SEQ ID NO:29).

In a further experiment the inventors investigated the consequences ofIL11RA1 knockout in this model of acute, chemically-induced kidneyinjury. Cre-lox mice that display fibroblast-specific knockout ofIL11RA1 in response to tamoxifen were treated with tamoxifen to deleteIL-11RA1 from col1A2-positive cells, and subsequently subjected tofolate-induced kidney injury as described above (or treatment withvehicle). After 21 days, collagen content was assessed using the HPAassay, and kidney function was determined by analysis of serum urealevels. The results are shown in FIG. 6 . The IL11RA1 knockout infibroblasts was found to protect the mice from folate-induced kidneyinjury, indicating a central role for IL-11 mediated signalling infibroblasts in the pathology of renal injury and consequent dysfunctionand secondary fibrosis.

In another experiment, mice were treated with folate as described above,and Enx203 was administered from day 3, at 10 mg/kg, biweekly. Bodyweight, kidney weight, renal collagen content, urine output and grosskidney morphology was evaluated after 28 days. The results are shown inFIG. 7 . At the end of the study, mice receiving anti-IL-11 antibodytherapy had increased kidney weights, less renal collagen, normal urineoutputs and improved gross morphology as compared to mice receiving IgGcontrol.

The inventors performed further experiments, in a chemically-inducedmodel of chronic kidney injury. Briefly, renal injury was induced inmice by folate treatment as described above, and were untreated for 21days. At 21 days post-induction of acute kidney injury by folatetreatment, chronic kidney disease had been established, as determined bya ˜2.5 fold increase in blood urea nitrogen (FIG. 8B), and a ˜2.9 foldincrease in the albumin to creatine ratio (ACR) in urine (FIG. 11 ,lower panel). The kidneys had lost ˜33% of their initial mass (FIG. 8C)and collagen levels were elevated ˜2.8-fold (FIG. 8A).

Mice were treated from day 21 with Enx203 or IgG control, at 10 mg/kg,biweekly. Mice were euthanised at the indicated day of the experiment,and renal collagen, serum urea levels, kidney weight and gross kidneymorphology were evaluated. The results are shown in FIG. 8 . Treatmentwith Enx203 was associated with reduced levels of renal collagen (FIG.8A) and reduced levels of serum urea, with an overall 51% reversal inBUN by week 12 of therapy (P<0.001 vs IgG; FIG. 8B).

Importantly, kidney weight increased in the Enx203-treated animals overtime, whilst renal collagen content decreased (FIGS. 8C and 8A), andserum urea levels decreased over time in Enx203-treated animals (FIG.8B). These results indicate that more than inhibiting folate-inducedrenal tissue injury responses, Enx203 treatment promoted regeneration offunctional kidney tissue, reversing the injury phenotype. The kidneys ofEnx203-treated mice harvested at days 84 and 105 also more closelyresembled healthy kidneys (FIG. 8D and FIG. 8E).

Histological analysis of renal sections from the mice revealed thatEnx203 treatment restored normal renal parenchyma (54% reversal inparenchymal loss by week 12 (P<0.001), cortical thickness and volume andcortex morphology in the kidneys of mice subjected to folate-inducedkidney injury (FIGS. 9 and 10 ).

The body weights of mice in this experiment were also monitoredthroughout the course of the experiment, and Enx203 treatment was foundto be associated with weight gain of folate treated mice towardscontrol, untreated levels (FIG. 11 , upper panel). The ACR in urine wasalso monitored in mice at days 21, 84 and 105 of the experiment, and theresults are shown in FIG. 11 (lower panel). Treatment with Enx203 wasfound to cause a substantial reduction in urinary ACR (˜50% reductionwithin 12 weeks) in folate-treated mice.

The results demonstrated that antagonism of IL-11-mediated signallingcould reverse renal fibrosis and produce extensive regeneration of thekidney parenchyma, which is associated with striking reversal of renalimpairment.

Example 3: Investigation of the Molecular Basis of theInhibition/Reversal of Renal Injury by Antagonists of IL-11 MediatedSignalling

The inventors undertook further investigations to evaluate the role ofIL-11 mediated signalling in renal tissue function and injury responses.

The transition of tubule epithelial cells to a mesenchymal cell-likephenotype is implicated in damage to the kidney parenchyma anddysfunction associated with renal injury (see e.g. Lovisa et al. Nat.Med. (2015) 21, 998-1009), and so the inventors investigated whetherIL-11 mediated signalling has a role in the epithelial-to-mesenchymaltransition for TECs.

Human primary tubule epithelial cells (TECs) were stimulated in vitrowith TGFB1, IL-11 (5 ng/ml, 24 h) or TGFB1+Enx203 (2 μg/ml, 24 h), andthe percentage of ACTA2^(+ve) cells and collagen expression wasevaluated using the Operetta high-content imaging platform.

The results are shown in FIG. 12 . Both of TGFB1 and IL-11 were found toincrease the proportion of ACTA2-expressing TECs, and to increasecollagen I expression by these cells. Treatment with Enx203 was found toreduce ACTA2 and collagen I expression by TECs in response toTGFB1/IL-11.

In separate experiments, human primary TECs were stimulated in vitro for24 hours with different concentrations of IL-11 (2.5 ng/ml, 5 ng/ml, 10ng/ml), TGFB1 (5 ng/ml), or TGFB1 (5 ng/ml)+Enx203 (2 ug/ml), andOperetta high-content imaging was used to visualise actin stress fibreformation using rhodamine-phalloidin (FIG. 13 , upper panels) andcollagen (FIG. 13 , lower panels).

As explained hereinabove, SNAIL has recently been shown to be a criticaldeterminant of TEC dysfunction following acute kidney injury. Itsexpression in TECs is associated with impaired TEC function andproliferation. Human primary TECs stimulated in vitro for 24 hours withdifferent concentrations of IL-11 (5 ng/ml), TGFB1 (5 ng/ml), TGFB1 (5ng/ml)+Enx203 (2 ug/ml) or TGFB1 (5 ng/ml)+IgG control (2 ug/ml) wereanalysed using the Operetta high-content imaging system for expressionof ACTA2 or SNAIL.

The results are shown in FIG. 14 . Treatment with IL-11 or TGFB1 wasfound to induce partial epithelial cell-mesenchymal cell transition(EMT), and expression of ACTA2 and SNAIL. However, the presence ofEnx203 strongly inhibited induction of ACTA2 and SNAIL.

Cell lysates were prepared from the cells, and analysed by western blotfor SNAIL expression. A separate western blot for GAPDH was performed asa protein loading control. The results are shown in FIG. 15 , anddemonstrate that SNAIL expression is upregulated in response to TGFB1and IL-11, and that TGFB-mediated upregulation of SNAIL was completelyprevented by the presence of Enx203.

Thus TGFB1-mediated induction of SNAIL was found to be IL-11-dependent.

The inventors investigated the expression of E-cadherin, the canonicalmarker of epithelial cell identity, by Western blots of proteins lysatesfrom kidney tissue of mice from the experiment described above in whichrenal injury was induced in mice by folate treatment as described above,and mice were subsequently treated from day 21 with Enx203 or IgGcontrol, at 10 mg/kg, biweekly.

The results are shown in FIG. 16 . Folate-induced renal injury was foundto reduce E-cadherin expression, whilst treatment with Enx203 was foundto restore E-cadherin expression.

Example 4: Analysis of the Effect of Inhibition of IL-11 MediatedSignalling in a Model of Physically-Induced Kidney Injury

A mouse model of acute renal injury was induced by unilateral uretericobstruction (UUO). Briefly, mice were treated by sham operation orureteric obstruction of one ureter.

Mice were treated with ENx203 (referred to as ‘3C6’ in FIG. 17B) or IgGcontrol antibody at day 4, 7 and 9 of treatment (20 mg/kg). The bodyweights of the mice were monitored throughout the experiment, andcollagen content in the kidney was assessed at 10 days post UUO surgeryusing the HPA assay.

The results are shown in FIG. 17 . Mice treated with Enx203 had reducedrenal collagen, and increased bodyweight as compared to mice subjectedto UUO and treated with IgG control antibody.

Example 5: Analysis of the Effect of Inhibition of IL-11 MediatedSignalling in a Model of Cisplatin-Induced Kidney Injury

Kidney injury was induced in 10 week old C57Bl/6J mice by administrationof 7 mg/kg cisplatin, once weekly for four consecutive weeks. A controlgroup was not administered with cisplatin.

Mice were administered biweekly from week 1 by IP injection with X203(antibody capable of binding to mouse IL-11 (and human IL-11) andinhibiting IL-11 mediated signalling) or an isotype-matched IgG controlantibody at a dose of 10 mg/kg, or with saline (control). Mice wereharvested for analysis after 8 weeks.

The amount of total collagen in the kidney was quantified on the basisof colourimetric detection of hydroxyproline using a Quickzyme TotalCollagen assay kit (Quickzyme Biosciences). The results are shown inFIG. 18B. Cisplatin treatment was associated with increased collagencontent of the kidney. Kidneys from cisplatin-treated mice administeredneutralising anti-IL-11 antibody had lower collagen content than kidneysfrom cisplatin-treated mice administered with IgG control antibody. Thekidneys of mice subjected to different treatments were also analysed forRNA expression of Col3, Fibronectin, MMP2, TIMP, CCL5 and CCL2 by qPCR.

Briefly, total RNA was extracted from snap-frozen kidney tissue usingTrizol (Invitrogen) and RNeasy Mini Kit (Qiagen). PCR amplificationswere performed using iScript cDNA Synthesis Kit (Biorad). Geneexpression was analyzed in duplicate by TaqMan (Applied Biosystems) orSYBR green (Qiagen) technology using StepOnePlus (Applied Biosystem)over 40 cycles. Expression data were normalized to GAPDH mRNA expressionand fold change was calculated relative to expression in saline-treatedcontrol subjects.

The results are shown in FIGS. 18C to 18H. Cisplatin treatment wasassociated with increased expression of Col3, Fibronectin, MMP2, TIMP,CCL5 and CCL2. Kidneys from cisplatin-treated mice administeredneutralising anti-IL-11 antibody tended to have lower expression ofCol3, Fibronectin, MMP2, TIMP, CCL5 and CCL2 as compared to kidneys fromcisplatin-treated mice administered with IgG control antibody.

1-32. (canceled)
 33. A method of inhibiting the transition of tubularepithelial cells (TECs) to a mesenchymal cell-like phenotype in asubject, comprising administering to a subject in need thereof aneffective amount of: (i) an anti-interleukin 11 (IL-11) antibody or anantigen-binding fragment thereof which is an antagonist ofIL-11-mediated signaling, or (ii) an anti-IL-11Rα antibody or anantigen-binding fragment thereof which is an antagonist ofIL-11-mediated signaling, thereby inhibiting the transition of TECs to amesenchymal cell-like phenotype in the subject.
 34. The method of claim33, wherein the subject is a subject suffering from impairment to renalfunction.
 35. The method of claim 33, wherein the subject is a subjectsuffering from kidney injury, or a disease or condition associated withkidney injury.
 36. The method of claim 33, wherein the subject is asubject suffering from a disease or condition characterised by damage toTECs.
 37. The method of claim 33, wherein the subject is a subjectsuffering from a disease or condition selected from: acute kidneyinjury, acute kidney failure, acute kidney disease, chronic kidneydisease, kidney damage, drug-induced kidney injury, ischemia-inducedkidney injury, tubular necrosis, acute tubular necrosis, autoimmunekidney injury and cancer.
 38. The method of claim 33, wherein the methodcomprises comprising administering to a subject in need thereof aneffective amount of an anti-IL-11 antibody or an antigen-bindingfragment thereof which is an antagonist of IL-11-mediated signaling. 39.The method of claim 38, wherein the anti-IL-11 antibody orantigen-binding fragment thereof comprises: (i) a heavy chain variable(VH) region incorporating the following CDRs: HC-CDR1 having the aminoacid sequence of SEQ ID NO:40 HC-CDR2 having the amino acid sequence ofSEQ ID NO:41 HC-CDR3 having the amino acid sequence of SEQ ID NO:42, ora variant thereof in which one or two or three amino acids in one ormore of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another aminoacid; and (ii) a light chain variable (VL) region incorporating thefollowing CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:43LC-CDR2 having the amino acid sequence of SEQ ID NO:44 LC-CDR3 havingthe amino acid sequence of SEQ ID NO:45, or a variant thereof in whichone or two or three amino acids in one or more of LC-CDR1, LC-CDR2, orLC-CDR3 are substituted with another amino acid.
 40. The method of claim38, wherein the anti-IL-11 antibody or antigen-binding fragment thereofcomprises: (i) a heavy chain variable (VH) region incorporating thefollowing CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:34HC-CDR2 having the amino acid sequence of SEQ ID NO:35 HC-CDR3 havingthe amino acid sequence of SEQ ID NO:36; and (ii) a light chain variable(VL) region incorporating the following CDRs: LC-CDR1 having the aminoacid sequence of SEQ ID NO:37 LC-CDR2 having the amino acid sequence ofSEQ ID NO:38 LC-CDR3 having the amino acid sequence of SEQ ID NO:39. 41.The method of claim 33, wherein the method comprises comprisingadministering to a subject in need thereof an effective amount of ananti-IL-11Rα antibody or an antigen-binding fragment thereof which is anantagonist of IL-11-mediated signaling.
 42. The method of claim 41,wherein the anti-IL-11Rα antibody or antigen-binding fragment thereofcomprises: (i) a heavy chain variable (VH) region incorporating thefollowing CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:46HC-CDR2 having the amino acid sequence of SEQ ID NO:47 HC-CDR3 havingthe amino acid sequence of SEQ ID NO:48; and (ii) a light chain variable(VL) region incorporating the following CDRs: LC-CDR1 having the aminoacid sequence of SEQ ID NO:49 LC-CDR2 having the amino acid sequence ofSEQ ID NO:50 LC-CDR3 having the amino acid sequence of SEQ ID NO:51. 43.The method of claim 33, wherein the subject is a subject in whichexpression of interleukin 11 (IL-11) or a receptor for IL-11 isupregulated.
 44. A method of inhibiting the transition of tubularepithelial cells (TECs) to a mesenchymal cell-like phenotype, comprisingcontacting TECs with: (i) an anti-IL-11 antibody or an antigen-bindingfragment thereof which is an antagonist of IL-11-mediated signaling, or(ii) an anti-IL-11Rα antibody or an antigen-binding fragment thereofwhich is an antagonist of IL-11-mediated signaling, thereby inhibitingthe transition of the TECs to a mesenchymal cell-like phenotype.
 45. Themethod of claim 44, wherein the method comprises comprisingadministering to a subject in need thereof an effective amount of ananti-IL-11 antibody or an antigen-binding fragment thereof which is anantagonist of IL-11-mediated signaling.
 46. The method of claim 45,wherein the anti-IL-11 antibody or antigen-binding fragment thereofcomprises: (i) a heavy chain variable (VH) region incorporating thefollowing CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:40HC-CDR2 having the amino acid sequence of SEQ ID NO:41 HC-CDR3 havingthe amino acid sequence of SEQ ID NO:42, or a variant thereof in whichone or two or three amino acids in one or more of HC-CDR1, HC-CDR2, orHC-CDR3 are substituted with another amino acid; and (ii) a light chainvariable (VL) region incorporating the following CDRs: LC-CDR1 havingthe amino acid sequence of SEQ ID NO:43 LC-CDR2 having the amino acidsequence of SEQ ID NO:44 LC-CDR3 having the amino acid sequence of SEQID NO:45, or a variant thereof in which one or two or three amino acidsin one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted withanother amino acid.
 47. The method of claim 45, wherein the anti-IL-11antibody or antigen-binding fragment thereof comprises: (i) a heavychain variable (VH) region incorporating the following CDRs: HC-CDR1having the amino acid sequence of SEQ ID NO:34 HC-CDR2 having the aminoacid sequence of SEQ ID NO:35 HC-CDR3 having the amino acid sequence ofSEQ ID NO:36; and (ii) a light chain variable (VL) region incorporatingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:37 LC-CDR2 having the amino acid sequence of SEQ ID NO:38 LC-CDR3having the amino acid sequence of SEQ ID NO:39.
 48. The method of claim44, wherein the method comprises comprising administering to a subjectin need thereof an effective amount of an anti-IL-11Rα antibody or anantigen-binding fragment thereof which is an antagonist ofIL-11-mediated signaling.
 49. The method of claim 48, wherein theanti-IL-11Rα antibody or antigen-binding fragment thereof comprises: (i)a heavy chain variable (VH) region incorporating the following CDRs:HC-CDR1 having the amino acid sequence of SEQ ID NO:46 HC-CDR2 havingthe amino acid sequence of SEQ ID NO:47 HC-CDR3 having the amino acidsequence of SEQ ID NO:48; and (ii) a light chain variable (VL) regionincorporating the following CDRs: LC-CDR1 having the amino acid sequenceof SEQ ID NO:49 LC-CDR2 having the amino acid sequence of SEQ ID NO:50LC-CDR3 having the amino acid sequence of SEQ ID NO:51.